NZ333802A

NZ333802A - Antitumor antisense sequences directed against R1 and R2 components of ribonucleotide reductase

Info

Publication number: NZ333802A
Application number: NZ333802A
Authority: NZ
Inventors: Jim A Wright; Aiping H Young
Original assignee: Genesense Technologies Inc
Priority date: 1996-08-02
Filing date: 1997-08-01
Publication date: 2001-09-28
Also published as: WO1998005769A2; DE69734589T2; EP0917569B1; IL128124A0; EP0917569A2; AU738592C; ATE309345T1; CN1231694A; CA2262776A1; DE69734589D1; ES2256893T3; AU3617597A; JP2000517167A; JP4301576B2; IL128124A; CN1189562C; US5998383A; WO1998005769A3; CA2262776C; AU738592B2

Abstract

An antisense oligonucleotide comprising at least seven to about thirty-five nucleotides and a sequence complimentary to a sequence of a mammalian ribonucleotide reductase gene, or an analogue thereof. The oligonucleotide is capable of inhibiting the proliferation of neoplastic cells. Also disclosed is a method of evaluating a compound for its ability to regulate a Ras signalling pathway by assaying for an agonist or antagonist of the interaction of R2 and Raf-1 and/or Rac-1. The method comprises providing a reaction mixture containing R2 and Raf-1 and/or Rac-1 under conditions which permit the interaction of R2 and Raf-1 and/or Rac-1, in the presence of a test compound; detecting the formation of complexes between R2 and Raf-1 and/or Rac-1 or activation of a Ras signalling pathway; and comparing to a control reaction in the absence of the test substance. Lower levels of complexes or activation in the reaction mixture indicate that the test compound interferes with the interaction of R2 and Raf-1 and/or Rac-1, and higher levels indicate that the test compound enhances the interaction of R2 and Raf-1 and/or Rac-1.

Description

<div class="application article clearfix" id="description"> WO 98/05769 PCT/CA97/00540 - 1 - TTTLE; ANTITUMOR ANTISENSE SEQUENCES DIRECTED AGAINST R1 AND R2 COMPONENTS OF RIBONUCLEOTIDE REDUCTASE This application claims benefit under 35 USC §119(e) of United States Provisional 5 Application Senal Number 60/023,040, filed August 2, 1996 and United States Provisional Application Serial Number 60/039,959, filed March 7, 1997 FIFI r> OF THE INVENTION The field of this invention relates to methods of controlling the tumorigenicity and/or metastasis of neoplastic cells Specifically it relates to the use of antisense sequences 10 directed against the R1 and R^components of ribonucleotide reductase BACKGROUND OETHfflNVENTION . r-«' The first unique step leading to DNA synthesis is the conversion of ribonucleotides to their corresponding deoxyribonucleotides, a reaction that is catalyzed in a cell cycle specific manner by the housekeeping gene ribonucleotide reductase [Lewis et al, 1978, Reichard, 1993, 15 Wright, 1989a; Wright et al, 1990a, Stubbe, 1989] The mammalian enzyme is composed of two dissimilar duneric protein components often called R1 and R2, which are encoded by two different genes located on different chromosomes [Bjorklund et al, 1993, Tonin et al, 1987] Mammalian protein R1 is a homodimenc structure, with a molecular weight of about 170 kDa, and has substrate sites and allosteric effector sites that control enzyme activity and substrate 20 specificity [Wright, 1989, Thelander et al , 1980, Caras et al, 1985; Wright et al, 1990a) Protein R2 is a homodimer, with a molecular weight of 88 kDa, and forms tw<o equivalent dinuclear iron centers that stabilizes a tyrosyl free radical required for catalysis [Wright et al, 1990a; Thelander et al, 1985, McClarty et al, 1990], R1 and R2 proteins interact at their C-temuna! ends to form an active holoenzyme {Reichard, 1993; Wright et al, 1990a, Davis et 25 al, 1994] R1 and R2 are differentially regulated during the cell cycle There is an S-phase correlated increase in the R2 protein resulting from its de novo synthesis [Lewis et al, 1978, Mann et al, 1988). The activity of ribonucleotide reductase, and therefore DNA synthesis and cell proliferation, is controlled in proliferating cells during the cell cycle by the synthesis and 30 degradation of the R2 component [Eriksson et al., 1984] The rate-limiting R2 component is a phosphoprotein capable of being phosphorylated by the CDC2 and CDK2 protein kinase mediators of cell cycle progression [Chan et al., 1993], and contains non-heme iron that stabilizes an unique tyrosyl free radical required for enzyme activity [Reichard, 1993, McClarty et al, 1990] 35 The levels of the R1 protein do not appear to change substantially during the cell cycle of proliferating cells and can be detected throughout the cell cycle Synthesis of R1 mRNA, like R2 mRNA appears to occur mainly during S phase [Eriksson et al, 1984, Choy et al., 1988; Mann et al, 1988] The broader distribution of the R1 protein during the cell cycle is attributed to its longer half life as compared to the R2 protein [Choy et al, 1988, Mann et al, Printed from Mimosa WO 98/05769 -2- PCT/CA97/00540 1988]. Regulation of ribonucleotide reductase, and particularly the R2 component, is altered in malignant cells exposed to tumor promoters or to the growth factor TGF-p [Amara, et al, 1994; Chen et al., 1993, Amara et al, 1995b, Hurta and Wright, 1995; Hurta et al., 1991] 5 Higher levels of enzyme activity have been observed in cultured malignant cells when compared to nonmalignant cells [Weber, 1983, Takeda and Weber, 1981, Wright e: al., 1989a), and increased levels of R2 protein and R2 mRNA have been found in pre-malignant and malignant tissues as compared to normal control tissue samples [Saeki et al, 1995, Jensen et al, 1994] 10 Regulation of ribonucleotide reductase, and in particular the R2 component, is elevated in transformed cells exposed to tumor promoters, or to transforming growth factor P in growth factor mediated mechanisms of tumor progression [Amara et al, 1996; Chen et al, 1993, Amara et al, 1995b] These studies are in tumor cells obtained from rodent and human tissues [Weber, 1983, Wright et al., 1989a, Saeki, et al, 1995; Jenson et al, 1994], and in cultured cells 15 selected for resistance to anti-tumor agents such as hydroxyurea [Lewis et al, 1973, Wright et al, 1989b). Compounds like hydroxyurea inhibit ribonucleotide reductase activity by destabilizing the iron center of the R2 protein causing the destruction of the tyrosyl free radical [McClarty et al, 1990], and preventing cells from progressing through S-phase of the 20 cell cycle [Ashihara and Baserga, 1979] Breakthroughs in molecular biology and the human genome project have opened previously unforeseen possibilities for targeted intervention with mammalian gene expression [Blaese, 1997, Feigner, 1997] These include approaches such as disruption of specific genes Antisense (AS) oligonucleotides (AS-ON) designed to hybridize with specific sequences 25 within a targeted mRNA are one example of such targeted intervention In general, antisense oligonucleotides interact well with phospholipid membranes [Akhter et al, 1991] Following their interaction with the cellular plasma membrane, they may be actively, or passively, transported into living cells [Loke et al., 1989], and this may occur by a saturable mechanism predicted to involve specific receptors [Yakubov et al., 1989]. 30 Many excellent reviews have covered the mam aspects of antisense technology and its enormous therapeutic potential. There are reviews on the chemical [Crooke, 1995], cellular [Wagner, 1994] and therapeutic [Hanania, et al, 1995, Scanlon, et al, 1995, Gewirtz, 1993] aspects of this rapidly developing technology Within a relatively short time, ample information has accumulated about the in iritro use of AS-ON in cultured primary cells and cell 35 lines as well as for in vivo administration of such ODNs for suppressing specific processes and changing body functions in a transient manner. Further, enough experience is now available in vttro and in vivo in animal models to predict human efficacy It would be useful to have antisense oligonucleotides available to control Printed from Mimosa > 33 WO 98/05769 PCT/CA97/00540 -3- tumorigenicity and/or metastatic potential in premalignant or malignant cells wherein the R1 and R2 components of ribonucleotide reductase were utilized. It is an object of the present invention to go some way towards fulfilling this desideratum and/or to provide the public with a useful choice. SUMMARY OF THE INVENTION 5 determine the malignant characteristics of cells. Altered R2 gene expression was found to cooperate with ras in mechanisms of malignant progression, and recombinant R2 expression resulted m increased membrane associated Raf-1 protein. These results suggest that R2 cooperates with Raf-1 and Rac-1 thereby affecting ras pathways and accordingly cell proliferation and in particular malignant progression. 10 The present inventors also showed that suppression of R2 gene expression reduced transformed properties of tumor cells. In particular, the present inventors demonstrated that novel R2 antisense decreased transformation. R1 antisense also suppressed transformed properties of tumor cells The R1 and R2 antisense are effective at low concentrations, and surprisingly normal cells were less sensitive to the antisense molecules 15 Aberrant expression of R2 was also found to result in increased resistance of tumor cells to chemotherapeutic agents R2 antisense decreased resistance of tumor cells to chemotherapeutic agents at concentrations of antisense that alone did not kill the neoplastic 20 modulating cell proliferation, preferably inhibiting the proliferation of tumor cells Compounds that may be used to modulate cell proliferation include inhibitors of ribonucleotide reductase expression i.e. inhibitors of transcription or translation of the gene encoding ribonucleotide reductase Antisense oligonucleotides complimentary to regions of the ribonucleotide reductase gene are particularly useful inhibitors. 25 In one embodiment, the present invention provides an antisense oligonucleotide having a sequence which is complementary to a nucleic acid sequence from a ribonucleotide reductase gene and comprises at least seven nucleotides or nucleotide analogues. In a preferred embodiment, the oligonucleotide is complementary to an mRNA region from a ribonucleotide reductase gene, more preferably the ribonucleotide reductase R1 or R2 gene 30 The invention also relates to a method of evaluating if a compound inhibits transcription or translation of a ribonucleotide reductase gene and thereby effects cell proliferation comprising transfectmg a cell with an expression vector comprising a recombinant molecule comprising a nucleic acid sequence encoding ribonucleotide reductase, and the necessary elements for the transcription or translation of the nucleic acid, administering a test 35 compound; and comparing the level of expression of the ribonucleotide reductase with the level obtained with a control in the absence of the test compound. Ras signalling pathway by assaying for an agonist or antagonist of the interaction of R2 and The present inventors have shown that aberrant expression of the R2 gene can cells. Broadly stated the present invention relates to compounds and methods for A method is also contemplated for evaluating a compound for its ability to regulate a ] WO 98/05769 PCT/CA97/00540 -4- Raf-1 and/or Rac-1 comprising providing a reaction mixture containing R2 and Raf-1 and/or Rac-1 under conditions which permit the interaction of R2 and Raf-1 and/or Rac-1, in the presence of a test compound; detecting the formation of complexes between R2 and Raf-1 and/or Rac-1 or activation of a Ras signalling pathway; and comparing to a control reaction in the absence of the test substance, wherein lower levels of complexes or activation in the reaction mixture indicate that the test compound interferes with the interaction of R2 and Raf-1 and/or Rac-1, and higher levels indicate that the test compound enhances the interaction of R2 and Raf-1 and/or Rac-1 The present invention also provides a pharmaceutical composition for modulating cell proliferation, preferably tumor cell proliferation, comprising at least one inhibitor of expression of R1 or R2, preferably an antisense oligonucleotide according to the present invention, or an analogue thereof, or a compound identified in accordance with a method of the invention, in admixture with a physiologically acceptable earner or diluent. Disclosed but not claimed is a method of modulating cell proliferation, preferably tumor ceil proliferation by contacting a cell with an effective amount of at least one compound that inhibits the expression of R2 or Rl, preferably an antisense oligonucleotide according to the present invention, or a compound identified in accordance with a method of the invention. Further disclosed is a method for reducmg cell proliferation, preferably tumor cell proliferation, comprising contacting a cell with an effective amount of an inhibitor of the expression of Rl or R2 preferably, antisense oligonucleotide according to the present invention, or a compound identified in accordance with a method of the invention. The present invention also provides a pharmaceutical composition for increasing the sensitivity of a tumor cell to a chemotherapeutic drug comprising at least one inhibitor of expression of Rl or R2, preferably an antisense oligonucleotide according to the present invention, or an analogue thereof, or a compound identified in accordance with a method of the invention, in admixture with a physiologically acceptable earner or diiuent. The present invention further provides a pharmaceutical composition for modulating the growth of a tumor cell that is resistant to a chemotherapeutic drug comprising at least one inhibitor of expression of Rl or R2, preferably an antisense oligonucleotide according to the present invention, or an analogue thereof, or a compound identified m accordance with a method of the invention, in admixture with a physiologically acceptable carrier or diluent The invention also contemplates the use of an antisense oligonucleotide according to the present invention, or a compound identified in accordance with a method of the invention, or an analogue thereof to prepare a medicament for modulating cell proliferation. DESCRIPTION OF THE DRAWINGS Other advantages of the present invention will be readily appreciated as the same becomes better understood by reference to the following detailed description when considered WO 98/05769 -5- PCT/CA97/00540 in connection with the accompanying drawings wherein. FIGURE 1A-C are photographs of gels (A and B) and two scans (C) showing the analysis of Myc-tagged R2 expression from stable mfectants by Western blot analysis using monoclonal anti-Myc epitope antibody 9E10 (A), polyclonal rabbit anti-R2 serum (B), and 5 during the cell cycle by flow cytometry [Blosmanis et al, 1987, Chadee et al, 1995], using antibody 9E10 (C) FIGURE 2A-C are photographs (A and B) and a graph (C) of experiments measuring transformed foci wherein (A) shows infection of BALB/c 3T3 (a) and NIH 3T3 (b) cells with SH/mR2 did not lead to focus formation <B) There was an increase in focus formation with 10 B3/mR2 (b) and N3/mR2 (d) compared to B3/SH (a) and N3/SH (c) after transfection with the T24 H-ras plasmid (C) The number of foci formed in three independent ras transfection experiments was plotted. FIGURE 3A-C are photographs of soft agar growth (A) and graphs (B and C) wherein (A) shows expression of Myc-R2 in ras-transformed cells resulted in an increased growth 15 efficiency in soft agar Examples shown are r-3/mR2 and uninfected r-3 cells (See Table 1). (B) Cl/mR2 cells showed reduced tumor latency and increased growth rate when compared to Cl/SH control cells where 3 x 105 cells from logarithmically growing cultures were collected and subcutaneously injected into five syngeneic C3H/HeN mice/cell line/experiment Results presented are from two independent experiments The p value of t test analysis of tumor 20 growth rates is shown, and indicates that the growth rates for the two cell lines are significantly different (C) CI /mR2 cells exhibited elevated metastatic potential. FIGURE 4A-C are graphs wherein (A) shows an increased amount of Raf-1 protein associated with the membrane in R2 overexpressing cells The recombinant R2 expressing cell lines B3/mR2, N3/mR2, CI /mR2, r-2/mR2, r-3/mR2 and NR4/mR2 (R2) were compared to their 25 respective control lines, B3/SH, N3/SH, Cl/SH, r-2/SH, r-3, and NR4 (control) In all cases, cells expressing recombinant R2 exhibited increased membrane associated Raf-1 protein, and when the two groups of cell lines were compared, they were found to be significantly different by t test analysis (p < 0.001) (B) Also shows an increase in the activity of mitogen activating protein kinase (MAPK-2) in R2 overexpressing cells. The recombinant R2 expressing lines 30 B3/mR2, N3/mR2,10T/mR2, Cl/mR2, r-2/mR2 and NR4/mR2 (R2) were compared to their respective control lines infected with LXSH (controls). In all cases tested, cells expressing recombinant R2 showed increased enzyme activity, and the difference between two groups was highly significant (p < 0.001). (C) Shows increased foci formation with N3/mR2 cells compared to N3/SH cells after transfection with the activated V12 Rac-1 plasmid [Jelinek et 35 al., 1994] The number of foci shown represents the average ± SE from two independent experiments. FIGURE 5A-B are photographs of gells showing examples of Southern blot analysis of CAD (A) and DHFR (B) DNA with mouse L cells. (A) H-4 cells not exposed to drug as a Printed from Mimosa WO 98/05769 -6- PCT/CA97/00540 control (a), H-4 cells from a colony that developed m the presence of 50 fiM PALA (b), or in the presence of 60 |xM PALA (c). DNA was digested to completion with Xbal (B) SC2 cells not exposed to drug as a control (a), SC2 cells from colonies that developed in the presence of 80 nM methotrexate (MTX) (b) and (c) DNA was digested to completion with Pstl. 5 FIGURE 6A-B are photographs of gells showing examples of Southern blot analysis of CAD (A) and DHFR (B) DNA with BALB/c 3T3 cells DNA was digested to completion with Pstl (A) B3/mR2 cells not exposed to PALA (a), and B3/mR2 cells from colonies that developed in the presence of 40'M PALA (b), or in the presence of 50|jM PALA (c). (B) B3/mR2 cells not exposed to MTX (a), and B3/mR2 cells from colonies that developed in the presence of 10 60 nM MTX (b), or in the presence of 80 nM MTX (c). Figure 7 is a photograph of a Western blot analysis of R2 protein levels in N/R2-4 (a) and N/R2+ASR2 (b) cells. To distinguish the vector R2 protein from the endogenous gene product in transfected cells, a human c-mvc epitope coding for ten amino acids plus methionine was placed at the 5' end of the cDNA for R2. Recombinant (upper band) and endogenous (lower 15 band) R2 protein is observed in lane a and is markedly reduced in R2 antisense containing cells (lane b). Both cell lines grew with approximately the same doubling time of about 16 hours. FIGURE 8 is a photograph of a gel showing p53-DNA binding activity in cells from colonies that developed in the presence of PALA, MTX or hydroxyurea (a) control IB cells that are p53-null, (b) B3/mR2 cells that grew in the presence of 20 (iM PALA, (c) B3/ R2c2 cells 20 that "grew in the presence of 40 ^iM PALA, (d) B3/mR2 cells that grew in the presence of 40 nM MTX, (e) B3/R2c2 cells that grew in the presence of 60 nM MTX, (f) B3/mR2 cells that grew in the presence of 0.20 mM hydroxyurea, and (g) B3/R2c2 cells that grew in the presence of 0.30 mM hydroxyurea Cells were incubated with 32P-labeled p53 consensus binding sequence in the presence of antibody 421, which activates p53 for DNA binding. Note the piesence of 25 complexes in all cell lines except in the IB control p53-null cells. Low molecular weight complex formation results from p53-DNA binding and high molecular weight complex formation results from antibody supershifted p53-DNA binding. FIGURE 9 is a graph showing the number of transformation foci in (a) NIH-3T3 mouse cells containing the H-ras oncogene, (b) NIH-3T3 mouse cells containing the H-ras oncogene and 30 the R2 antisense sequence and (c) NIH-3T3 mouse cells containing the H-ras oncogene and the coding region sequence for R2 Results are averages of three experiments. Figure 10A-B are photographs of a Western blot analysis of AS-1I-626-20 inhibition (A) and inhibition by a variety of R2 antisense oligonucleotides (B) of ribonucleotide reductase R2 protein level m L60 mouse tumor cells 35 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 1. Antisense and Rtbozymes The present invention provides compounds that inhibit the expression of a ribonucleotide reductase protein and thereby modulate cell proliferation The compounds may Printed from Mimosa ^«3o8'02 WO 98/05769 PCT/CA97/00540 -7- mhibit the expression of the ribonucleotide reductase by inhibiting the transcription of the gene, or the translation of the mRNA to protein. Such compounds may mdude antisense oligonucleotides and nbozvmes. The term "antisense oligonucleotide" as used herein means a nucleotide sequence that 5 is complementary to its target. The term "oligonucleotide" refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages. The term also includes modified or substituted oligomers comprising non-naturally occurring monomers or portions thereof, which function similarly. Such modified or substituted 10 oligonucleotides may be preferred over naturally occurring forms because of properties such as enhanced cellular uptake, or increased stability m the presence of nucleases The term also includes chimeric oligonucleotides which contain two or more chemically distinct regions. For example, chimeric oligonucleotides may contain at least one region of modified nucleotides that confer beneficial properties (e.g. mcreased nuclease resistance, increased uptake into 15 cells), or two or more oligonucleotides of the invention may be joined to form a chimeric oligonucleotide. The antisense oligonucleotides of the present invention may be ribonucleic or deoxyribonucleic aads and may contain naturally occurring bases including adenine, guanine, cytosme, thymidine and uracil. The oligonucleotides may also contain modified bases such as 20 xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosme, 6-aza uracil, 6-aza cytosme and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenmes, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other 25 aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5-trifluoro cytosme. Other antisense oligonucleotides of the invention may contain modified phosphorous, oxygen heteroatoms m the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. For example, the 30 antisense oligonucleotides may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates. In an embodiment of the invention there are phosphorothioate bonds links between the four to six 3'-terminus bases. In another embodiment phosphorothioate bonds link all the nucleotides. The antisense oligonucleotides of the invention may also comprise nucleotide analogs 35 that may be better suited as therapeutic or experimental reagents. An example of an oligonucleotide analogue is a peptide nucleic acid (PNA) wherem the deoxynbose (or nbose) phosphate backbone in the DNA (or RNA), is replaced with a polyanude backbone which is similar to that found in peptides (P.E. Nielsen, et al Science 1991, 254, 1497). PNA analogues WO 98/05769 -8- PCT/CA97/tfe540 have been shown to be resistant to degradation by enzymes and to have extended lives m vivo and in vitro. PNAs also bind stronger to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand. Other oligonucleotides may contain nucleotides containing polymer backbones, cyclic backbones, or acvclic backbones. For 5 example, the nucleotides may have morpholino backbone structures (U.S. Pat. Nol 5,034, 506) Oligonucleotides may also contain groups such as reporter groups, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an antisense oligonucleotide. Antisense oligonucleotides may also have sugar mimetics. 10 The antisense oligonucleotides may be selected such that they exhibit the least likelihood of dimer formation, self-complementary interactions, and binding potential to the ribonucleotide reductase mRNA other than target sequence. These properties may be determined using the computer modeling program OLIGO Primer Analysis software Version 3 4 (National Biosciences). The program allows the determination of a qualitative estimation of 15 these three parameters and indicates "no potential", "some potential", or "essentially complete potential" Oligonucleotides are preferably selected that have estimates of "some potential" or "no potential", most preferably "no potential", m all three parameters as described in Tables 7 and 11. The oligonucleotides are also selected so that their function is not substantially affected by any modifications or substitutions. 20 The antisense oligonucleotides of the present invention are preferably complementary to the mRNA region from the ribonucleotide reductase gene. More preferably, the antisense oligonucleotide is complementary to an mRNA region from the ribonucleotide reductase R2 gene. The antisense oligonucleotides generally comprise at least seven nucleotides or 25 nucleotides analogs, more preferably, at least 20 nucleotides or nucleotide analogs, most preferably 30-35 nucleotides or nucleotide analogs The sequences of preferred antisense oligonucleotides according to the present invention can be found in Tables 11 and 7 and are SEQ ID. NOS. 1-102 and SEQ. ID. NOS 103-161. More preferred oligonucleotides are shown in Table 12. Most preferred oligonucleotides have the SEQ.ID.NOS 1, 2, 12, 16, 18, 21, 25, 29, 34, 30 42,44, 45, 46, 52, 53, 59, 60, 64, 65, 66, 68, 69, 70, 72, 73, 74, 76, 78, 79, 80. 90, 91, 92, 96, 99, 100 and 102 as shown in Table 7 The antisense oligonucleotides of the invention may be prepared by conventional and well-known techniques. For example, the oligonucleotides may be prepared using solid-phase synthesis and in particular usmg commercially available equipment such as the equipment 35 available from Applied Biosystems. It is also preferred to substantially purify the oligonucleotides so that they are free of any other factors which would interfere with their activity Oligonucleotides of the invention may also be identified using genetic complementation techniques, or usmg the probes described herein It is also well within the WO 98/05769 -9- PCT/CA97/00540 skill in the art to prepare modified or substituted antisense oligonucleotides A ribozyme sequence may also be used to modulate cell proliferation The nbozyme has homologous or complementary sequences to an antisense oligonucleotide of the invention and the necessary catalytic centre for cleaving the oligonucleotide The ribozyme type utilized 5 in the present invention may be selected from types known in the art Several ribozyme structural families have been identified including Group I mtrons, RNase P, the hepatitis delta virus nbozyme, hammerhead nbozymes, and the hairpin ribozyme originally derived from the negative strand of the tobacco ringspot virus satellite RNA (sTRSV) (Sullivan, 1994, U.S Patent No. 5,225,347, columns 4 to 5). The latter two families are derived from viroids and 10 virusoids, in which the ribozyme is believed to separate monomers form oligomers created during rolling circle replication (Symons, 1989 and 1992). Hammerhead and hairpin ribozyme motifs are most commonly adapted for trans-cleavage of mRNAs for gene therapy (Sullivan, 1994) Hairpin nbozymes which are presently in clinical trials are preferably used m the present invention In general the ribozyme is from 30 to 100 nucleotides in length 15 2. Methods for Evaluating Compounds The invention contemplates a method of evaluating if a compound inhibits transcnption or translation of a ribonucleotide reductase gene and thereby modulates (1 e. reduces) cell proliferation comprising transfecting a cell with an expression vector compnsing a nucleic acid sequence encoding ribonucleotide reductase, the necessary elements for the 20 transcription or translation of the nucleic acid; administering a test compound, and comparing the level of expression of the ribonucleotide reductase with the level obtained with a control in the absence of the test compound An expression vector comprising a nucleic acid sequence encoding ribonucleotide reductase may be constructed having regard to the sequence of the gene using procedures known 25 in the art Suitable transcription and translation elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropnate elements is dependent on the host cell chosen, and may be readily accomplished by one of ordinary skill in the art Examples of reporter genes are genes encoding a protein such as [J-galactosidase (e g 30 lacZ), chloramphenicol, acetyl-transferase, firefly luciferase, or an immunoglobulin or portion thereof. Transcription of the reporter gene is monitored by changes in the concentration of the reporter proteins such as (i-galactosidase etc. This makes it possible to visualize and assay for expression of recombinant molecules to determine the effect of a substance on expression of the ribonucleotide reductase gene 35 Host cells suitable for carrying out the present invention include CHO, COS, BHK, 293 and HeLa Protocols for the transfection of mammalian cells are well known in the art and include calcium phosphate mediated electroporation, and retroviral, and protoplast fusion-mediated transfection Printed from Mimosa WO 98/05769 -10- PCT/CA97/00540 The present inventors have found that R2 interacts or cooperates with Raf-1 and/or Rac-1 thereby affecting the Ras signalling pathways Therefore, the invention also contemplates a method for evaluating a compound for its ability to regulate a Ras signalling pathway by assaying for an agonist or antagonist (1 e stimulator or inhibitor) of the 5 interaction of R2 and Raf-1 and/or Rac-1 The basic method for evaluating if a compound is an agonist or antagonist of the interaction of R2 and Raf-1 and/or Rac-1, is to prepare a reaction mixture containing R2 and Raf-1 and/or Rac-1 under conditions which permit the interaction of R2 and Raf-1 and/or Rac-1, in the presence of a test compound The test compound may be initially added to the mixture, or may be added subsequent to the addition of the R2 and Raf-1 10 and/or Rac-1. Control reaction mixtures without the test compound or with a placebo are also prepared. The formation of complexes or activation of the pathway is detected and the formation of complexes or activation of the pathway m the control reaction but not in the reaction mixture indicates that the test compound interferes with the interaction of R2 and Raf-1 and/or Rac-1. The reactions may be carried out in the liquid phase or R2 and Raf-1 15 and/or Rac-1, or test compound may be immobilized The invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of the interaction of R2 and Raf-1 and/or Rac-1 Thus, the invention may be used to assay for a compound that competes for the same binding site of R2. The invention also contemplates methods for identifying compounds that bmd to 20 proteins that interact with R2 and thereby inhibit R2 Protein-protein interactions may be identified using conventional methods such as co-immunoprecipitation, crosshnkmg and co-punfication through gradients or chromatographic columns Methods may also be employed that result m the simultaneous identification of genes which encode proteins interacting with R2 These methods include probing expression libraries with labeled R2. 25 Two-hybrid systems may also be used to detect protein interactions in vivo Generally, plasmids are constructed that encode two hybrid proteins A first hybrid protein consists of the DNA-binding domain of a transcription activator protein fused to R2, and the second hybrid protein consists of the transcription activator protem's activator domain fused to an unknown protein encoded by a cDNA which has been recombined into the plasmid as part of 30 a cDNA library The plasmids are transformed into a strain of yeast (e.g. S. cemrisiae) that contains a reporter gene (e.g lacZ, luciferase, alkaline phosphatase, horseradish peroxidase) whose regulatory region contains the transcription activator's bindmg site The hybrid proteins alone cannot activate the transcription of the reporter gene. However, interaction of the two hybrid proteins reconstitutes the functional activator protein and results in expression 35 of the reporter gene, which is detected by an assay for the reporter gene product It will be appreciated that fusion proteins may be used in the above-described methods. In particular, R2 fused to a glutathione-S-transferase may be used in the methods. The compounds identified usmg the method of the invention include but are not Printed from Mimosa WO 98/05769 -11- PCT/CA97/00540 limited to peptides such as soluble peptides including Ig-tailed fusion peptides, members of random peptide libraries and combinatorial chemistry-derived molecular libraries made of D-and/or L-configuration ammo acids, phosphopeptides ( including members of random or partially degenerate, directed phosphopeptide libraries), antibodies (e g polyclonal, 5 monoclonal, humanized, anti-idiotypic, chimeric, single chain antibodies, fragments, (e.g Fab, F(ab)2, and Fab expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules. The compound may be an endogenous physiological compound or it may be a natural or synthetic compound The reagents suitable for applying the methods of the invention to evaluate 10 compounds that modulate R2 and Raf-1 and/or Rac-1 interactions may be packaged into convenient kits providing the necessary materials packaged into suitable contamers The kits may also include suitable supports useful in performing the methods of the invention The compounds identified usmg the methods described herein, and other inhibitors of R2 expression described herein (e g. antisense to R2) may be used for modulating a Ras 15 pathway In particular, the compounds may be used to inhibit the signal-transduction properties of Raf-1 and/or Rac-1, inhibit cell proliferation, alter the cell cycle, and downregulate the immune response in patients with autoimmune diseases. In an embodiment of the invention, the compounds have anti-oncogene or tumor suppressor activity 3. Methods and Compostttons for Modulating Cell GrowthlMetastasts 20 The antisense oligonucleotides, nbozymes, and compounds identified using the methods of the invention modulate cell proliferation and m particular tumor cell proliferation Therefore, methods are provided for interfering with cell proliferation, preferably tumor cell proliferation compnsmg contacting tissues or cells with one or more of antisense oligonucleotides, nbozymes, and compounds identified usmg the methods of the 25 invention. Preferably, an antisense oligonucleotide as shown in Table 7 or 11 or 12 is administered. The term "contact" refers to the addition of an antisense oligonucleotide, ribozyme etc, m a liquid carrier to a cell suspension or tissue sample, or to administering the oligonucleotides etc. directly or indirectly to cells or tissues within an animal 30 The methods may be used to treat proliferative disorders including various forms of cancer such as leukemias, lymphomas (Hodgkms and non-Hodgkins), sarcomas, melanomas, adenomas, carcinomas of solid tissue, hypoxic tumors, squamous cell carcinomas of the mouth, throat, larynx, and lung, genitourinary cancers such as cervical and bladder cancer, hematopoietic cancers, colon cancer, breast cancer, pancreatic cancer, head and neck cancers, 35 and nervous system cancers, benign lesions such as papillomas, arthrosclerosis, psoriasis, primary and secondary polythemia, mastocytosis, autoimmune diseases, angiogenesis, bacterial infections, and viral infections, such as HIV infections, hepatitis or herpes infections Printed from Mimosa WO 98/05769 -12- PCT/CA97/00540 The antisense oligonucleotides, ribozymes,and compounds identified using the methods of the invention may also be used to treat drug resistant tumors Examples of drug resistant tumors are tumors resistant to hydroxyurea; tumors expressing high levels of P-glycoprotein which is known to confer resistance to multiple anticancer drugs such as 5 colchicine, vinblastine and doxorubicin; or, tumors expressing the multi-drug resistance protein as described in R. Deeley et al., Science, 258 1650-1654,1992 Antisense oligonucleotides of the invention have also been found to reduce metastasis In an embodiment of the invention, a method is provided for reducing metastasis m a subject comprising administering an amount of an antisense oligonucleotide of the invention effective 10 to reduce metastasis Preferably the antisense oligonucleotide has the sequence shown in SEQ ID. NOS. 1-102 or SEQ. ID NOS. 103-161, most preferably a sequence shown in Table 12 Selected antisense oligonucleotides, nbozymes, and compounds may be tested for their ability to modulate cell growth and in particular tumor cell growth, or to reduce metastasis in vitro and in vivo systems as described herein 15 For therapeutic applications, the antisense oligonucleotides, nbozymes, and compounds identified using the methods of the mvention may be formulated into pharmaceutical compositions. The pharmaceutical compositions may comprise one or more antisense oligonucleotides, ribozymes, and compounds identified using the methods of the invention for adminstration to subjects in a biologically compatible form suitable for 20 administration to a subject. The compositions of the invention can be intended for administration to humans and vanous other mammals, such as ovines, bovines, equines, swine, canines, and felines The pharmaceutical compositions of the invention may be administered in different ways depending upon whether local or systemic treatment is desired, and upon the area to be 25 treated. The compositions can be administered orally, subcutaneously or parenterallv including intravenous, intraarterial, intramuscular, intraperitoneally, and intranasal administration as well as intrathecal and infusion techniques as required by the malignant cells being treated For delivery within the CNS intrathecal delivery can be used with for example an Ommaya reservoir or other methods known in the art The pharmaceutical^ acceptable carriers, 30 diluents, adjuvants and vehicles as well as implant carriers generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating material not reacting with the active ingredients of the mvention. Cationic lipids (e.g. Lipofectin, Life Technologies) may also be included in the composition to facilitate oligonucleotide uptake Implants of the compounds are also useful. In general the pharmaceutical compositions are sterile 35 The antisense oligonucleotides and nbozymes of the invention may be delivered using viral or non-viral vectors Sequences may be incorporated into cassettes or constructs such that an antisense oligonucleotide or ribozyme of the invention is expressed in a cell. Generally the construct contains the proper transcriptional control region to allow the oligonucleotide or Printed from Mimosa WO 98/05769 -13- PCT/CA97/00540 antisense oligonucleotide to be transcribed in the cell Therefore, the invention provides vectors comprising a transcription control sequence operabvely linked to a sequence which encodes art antisense oligonucleotide or nbozyme of the invention. The present invention further provides host cells, selected from suitable eucaryotic 5 and procaryotic cells, which are transformed with these vectors Such transformed cells allow the study of the function and the regulation of malignancy and the treatments of the present invention Vectors are known or can be constructed by those skilled in the art and should contain all expression elements necessary to achieve the desired transcnption of the sequences Other 10 beneficial characteristics can also be contained within the vectors such as mechanisms for recovery of the nucleic acids in a different form. Phagemids are a specific example of such beneficial vectors because they can be used either as plasmids or as bacteriophage vectors. Examples of other vectors include viruses such as bacteriophages, baculoviruses and retroviruses, DNA viruses, liposomes and other recombination vectors The vectors can also 15 contain elements for use in either procaryotic or eucaryotic host systems One of ordinary skill in the art will know which host systems are compatible with a particular vector The vectors can be introduced into cells or tissues by any one of a vanety of known methods within the art. Such methods can be found generally described in Sambrook et al, Molecular Cloning. A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 20 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang et al, Somatic Gene Therapy, CRC Press, Ann Arbor, MI (1995), Vega et al. Gene Targeting, CRC Press, Ann Arbor, MI (1995), Vectors. A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA (1988) and Gilboa et al (1986) and include, for example, stable or transient transfection, hpofection, electroporation 25 and infection with recombinant viral vectors. Introduction of nucleic acids by infection offers several advantages. Higher efficiency can be obtained due to their infectious nature Moreover, viruses are very specialized and typically infect and propagate in specific cell types Thus, their natural specificity can be used to target the vectors to specific cell types in vivo or within a tissue or 30 mixed culture of cells. Viral vectors can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events Additional features can be added to the vector to ensure its safety and/or enhance its therapeutic efficacy Such features include, for example, markers that can be used to negatively select against cells infected with the recombinant virus An example of such a 35 negative selection marker is the TK gene that confers sensitivity to the anti-viral gancyclovir Negative selection is therefore a means by which infection can be controlled because it provides inducible suicide through the addition of antibiotic. Such protection ensures that if, for example, mutations arise that produce altered forms of the viral vector or sequence, cellular Printed from Mimosa WO 98/05769 -14- PCT/CA97/00540 transformation will not occur Features that limit expression to particular cell types can also be included Such features include, for example, promoter and regulatory elements that are specific for the desired cell type. Recombinant viral vectors are another example of vectors useful for m vivo 5 introduction of a desired nucleic acid because they offer advantages such as lateral infection and targeting specificity Lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. The result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. This is in contrast to 10 vertical-type of infection in which the infectious agent spreads only through daughter progeny. Viral vectors can also be produced that are unable to spread laterally This characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells A vector to be used in the methods of the invention may be selected depending on the 15 desired cell type to be targeted For example, if breast cancer is to be treated, then a vector specific for such epithelial cells should be used. Similarly, if cells of the hematopoietic system are to be treated, then a viral vector that is specific for blood cells and their precursors, preferably for the specific type of hematopoietic cell, should be used. Retroviral vectors can be constructed to function either as infectious particles or to 20 undergo only a single initial round of infection. In the former case, the genome of the virus is modified so that it maintains all the necessary genes, regulatory sequences and packaging signals to synthesize new viral proteins and RNA. Once these molecules are synthesized, the host cell packages the RNA into new viral particles which are capable of undergoing further rounds of infection The vector's genome is also engineered to encode and express the desired 25 recombinant gene In the case of non-infectious viral vectors, the vector genome is usually mutated to destroy the viral packaging signal that is required to encapsulate the RNA into viral particles Without such a signal, any particles that are formed will not contain a genome and therefore cannot proceed through subsequent rounds of infection The specific type of vector will depend upon the intended application The actual vectors are also known and readdy 30 available within the art or can be constructed by one skilled m the art using well-known methodology. If viral vectors are used, for example, the procedure can take advantage of their target specificity and consequently, do not have to be administered locally at the diseased site. However, local administration may provide a quicker and more effective treatment, 35 administration can also be performed by, for example, intravenous or subcutaneous injection into the subject. Injection of the viral vectors into a spinal fluid can also be used as a mode of administration. Following injection, the viral vectors will circulate until they recognize host cells with the appropriate target specificity for infection. Printed from Mimosa WO 98/05769 -15- PCT/CA97/00540 Transfection vehicles such as liposomes can also be used to introduce the non-viral vectors described above into recipient cells -within the inoculated area Such transfection vehicles are known by one skilled within the art The pharmaceutical compositions and vectors of the invention may be administered 5 in combination with other drugs or singly, consistent with good medical practice and treatment modalities that are known in the art Examples of other drugs which may be administered in combination with the compositions etc. of the invention are cytotoxic agents, immunotoxins, alkylating agents, anti-metabolites, antitumor antibiotics and other anti-cancer drugs Dosing of the antisense oligonucleotides, ribozymes, and compounds will depend on 10 the severity and responsiveness of the condition to be treated with a course of treatment lasting from several days to several months or until diminution of the disease is achieved. Optimal dosing schedules may be calculated using measurements of drug accumulation m the body Persons of ordinary skill m the art can readily determine optimum dosages, dosing methodologies, and repetition rates Optimum dosages may vary depending on the relative 15 potency of individual oligonucleotides, and can generally be determined based on EDggS in in vitro and in vivo animal studies The pharmaceutical compositions or vectors of the invention, and combination drugs may each be administered at non-cytotoxic or cytotoxic doses, or one may be administered at a cytotoxic dose and the other at a non-cytotoxic dose The doses may be selected to provide a synergistic effect 20 EXAMPLES The examples provide an analysis of malignancy related characteristics of cells containing deregulated R2 expression achieved by gene transfer techniques Overexpression of R2 leads to an increased frequency of transformed foci formation by mouse fibroblasts following transfection with activated H-ras In addition, expression of recobminant R2 in ras-25 transformed cells resulted in enhanced colony forming efficiency in soft agar, and markedly elevated tumorigenic and metastatic potential in vivo Furthermore, deregulated R2 expression can cooperate with other oncogenes like rac-1 in mechanisms of transformation The results herein demonstrate for the first time that the R2 component of mammalian ribonucleotide reductase is a malignancy determinant that can synergize with 30 activated oncogenes to modify malignant potential, and supports a model in which these effects are mediated through alterations in major Ras pathways that are brought about by deregulated R2 gene expression The observations presented here indicated that R2 can also participate in other critical cellular functions, and can play a direct role in determining malignant potential through oncogene cooperahvity. 35 The examples further demonstrate that ribonucleotide reductase R2 gene expression can play a significant role in determining drug sensitivity characteristics, and that this appeas to occur at least in part through a mechanism involving genomic instability The mechanism through which aberrant R2 expression modifies drug sensitivities Printed from Mimosa WO 98/05769 -16- PCT/CA97/00540 does not appear to require the direct involvement of p53 mutation or loss of wild type p53 function, although it is possible that genetic events downstream of a p53 regulated pathway are involved As shown in Example 1 a relationship exists between increased R2 expression and activation of a ras pathway involving the Raf-1 protein and mitogen-activated protein 5 kinase-2 (MAPK) activity. Recombinant R2 gene expression in Balb/c 3T3 and NIH-3T3 cells significantly increases both Raf-2 protein activation and mitogen-activating protein kinase (MAPK) activity. A hypothesis for the above obserations can be made, but it is not to be construed as limiting the present invention to this one mode of action These observations imply that the 10 R2 protein is capable of acting as a signal molecule in the MAPK pathway, in addition to its role as a rate-limiting component of ribonucleotide reduction Transcription factors like the product of the c-myc gene are downstream targets of the MAPK pathway, and control for example, expression of cyclins A, D and E, which are important in the regulation of checkpoints during cell cycle progression [Hunter, 1994; 1995]. Compromising cell cycle 15 checkpoint controls enhance genomic destabilization and facilitates DNA amplification [Kohn, 1996; Livingston et al., 1992], c-myc overexpression has also been directly linked to gene amplification mechanisms involving DHFR [Mai, 1994] These obserations suggest that alterations in thjtf-MAPK pathway through aberrant R2 expression may be at least partly responsible for the observed changes in drug sensitivities and genomic integrity 20 Example 3 demonstrates that short antisense sequences directed against the Rl and R2 components have anti-tumor activity and are cytotoxic to the neoplastic cells Further, the R2 antisense sequences can also act synergistically with well known chemotherapeutic agents Very low concentrations (non-toxic) of short antisense sequences reduced the resistance of the neoplastic cells to chemotherapeutic agents such as N-(phosphonacetyl)-L-aspartate (PALA) 25 and methotrexate (MTX) as well as hydroxyurea As shown in the Example, cells were transfected with a vector containing the R2 sequence in an antisense oritentation These cells were more sensitive to the chemotherapeutic agents Also, mouse IOTV2 cells which are drug resistant, when transfected with R2 sequence in the antisense orientation, were found to have significantly reduced resistance (increased sensitivity) to the chemotherapeutic agents Short 30 synthetic antisense sequence complementary to the R2 sequence also provided increases sensitivity. The above discussion provides a factual basis for the use of antisense oligonucleotides and nbozymes directed against the R2 mRNA The methods used with and the utility of the present invention can be shown by the following non-limiting examples and accompanying 35 figures GENERAL METHODS: GENERAL METHODS IN MOT.RCULAR BIOLOGY' Standard molecular biology techniques known in the art and not specifically described were generally followed as in Sambrook et al, Printed from Mimosa WO 98/05769 -17- PCT/CA97/00540 Molecular Cloning• A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); in Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989); and in Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York (1988) Polymerase chain reaction (PCR) was carried out generally as 5 m PCR Protocols A Guide To Methods And Applications, Academic Press, San Diego, CA (1990) Vectors can be constructed for the present invention by those skilled in the art and should contain all expression elements necessary to achieve the desired transcription of the sequences The expression elements can be selected to allow expression only in the cell being 10 targeted Other beneficial characteristics can also be contained within the vectors such as mechanisms for recovery of the nucleic acids in a different form. One of ordinary skill in the art will know which expression elements are compatible with a particular cell type. The vectors can be introduced into cells or tissues by any one of a variety of known methods within the art as described herein above 15 GENERAL MLlHODS IN IMMUNOLOGY Standard methods in immunology known in the art and not specifically described were generally followed as in States et al (eds), Basic and Clinical Immunology (8th Edition), Appleton & Lange, Norwalk, CT (1994) and Mishell and Shngi (eds), Selected Methods in Cellular Immunology, W.H Freeman and Co, New York (1980) 20 ASSAYS FOR TUMORIGENICITY AND METASTASIS Malignancy potential was determined as reported previously [Wright, 1989a; Egan et al., 1987a, 1987b; Damen et al, 1989, Taylor et al., 1992; Stokoe et al, 1994}. Six to eight week old C3H/HeN syngeneic mice (Charles River, Quebec) were used to evaluate tumongenic and metastatic potential of the cells Cells were prepared from subconfluent, logarithmically growing cultures, collected by 25 gentle treatment with trypsin/EDTA solution and adjusted to appropnate concentration in a balanced salt solution For the tumorigenicity (tumor latency) assay, 1 x 10s cells in a 0 1 ml volume were injected subcutaneously into the back of mice and the tune required to form a tumor (2X2 mm) detectable by palpation was recorded. The growth of tumors was also evaluated by measuring 30 tumor diameters, and estimating tumor base area each day following tumor appearance [Damen et al., 1989]. Tumor size was determined by multiplying the dimensions of the cross-section of the tumor. Tumors were removed from the mice and tumor weight was recorded 21 days later In the case of no tumor formation, mice were kept for 2 months after injection and then sacrificed. 35 For experimental metastasis assays (determination of metastatic potential), 1 x 105 cells in a 0.2 ml volume were injected into the tail veins of 6-8 week old C3H/HeN syngeneic mice and an estimate of the number of lung tumors was made 21 days later The mice were sacrificed, and the lungs were stained by injecting Bourn's solution (picnc acid, formaldehyde, Printed from Mimosa WO 98/05769 -18- PCT/CA97/00540 acehc acid (15 5 1)) intratracheal^ [Egan et al., 1987b; Damen et al, 1989] Pulmonary tumors were counted with the aid of a dissecting microscope To confirm that equal numbers of test and control cells were injected, duplicate culture plates containing growth medium were inoculated with 100 cells per plate. After 10 days in culture, plates were stained with methylene blue and 5 colonies were scored. RIBONUCLEOTIDE REDUCTASE ASSAY- Ribonucleotide reductase activity m crude extracts prepared from cells is assayed as previously described [Lewis et al, 1978, Hurta and Wright, 1992; Hurta et al, 1995] Enzyme preparations are obtained from logarithmically growing cells lysed in phosphate buffered saline, pH 7 2, containing 1 mM dithiothreitol and 1 10 mM protease inhibitor, AEBSF (Calbiochem, San Francisco, CA), by three cycles of freeze-thawing. Following centrifugabon, the supernatant is used for enzyme activity assays with [14C]-CDP (Moravek Biomedical, Brea, CA), as detailed previously [Lewis et al, 1978, Hurta and Wright, 1992, Fein et al, 1996, Choy et al, 1988] WESTERN BLOT ANALYSIS. The procedures used have been reported [Fan et al, 1996a; 15 1996b, Choy et al, 1988]. Briefly, following cell extract preparation, total protein content was determined, and an aliquot was analyzed on 10% linear SDS-polyacrylamide gel After protein transfer and blocking, membranes were incubated with anti-R2 rabbit polyclonal antibody Alkaline phosphatase conjugated goat anti-rabbit IgG (Sigma) was used for protein R2 detection 20 EXAMPLE 1 R2 COOPERATES WITH ACTIVATED ONCOGENES To determine the malignant potential of deregulated expression of the rate-limiting R2 component of ribonucleotide reductase, the properties of cells stably infected with a retroviral expression vector (SH/mR2) carrying the R2 component [Fan et al, 1996b], were 25 investigated. Further the interaction between R2 and activated oncogenes was explored MATERIALS AND METHODS Expression Vectors: The retroviral expression vector for the human Myc epitope-tagged mouse R2 component, SH/mR2, was constructed and packaged as described in Fan et al [1996b] The infectivity of the viral stock was >1 x 104 colony-forming units/ml. Plasmid pH06Ti which 30 expresses T-24 H-ras and a selective marker neo was used for malignant transformation [Egan et al., 1987a, 1987b; Taylor et al., 1992]. The activated Rac-1 plasmid (V12 Rac-1) was kindly provided by M Symons [Stokoe, et al., 1994] Cells and Cell Culture• The mouse cell lines, BALB/c 3T3, NIH 3T3, four lines of T24 H-ras transformed lOTVj cells, named CI, NR4, r-2 and r-3 have been previously used as recipients of 35 the R2 retroviral vector [Fan et al, 1996b] Cells were routinely cultured in a-mimmal essential medium (a-MEM)(Gibco, Grand bland, NY) supplemented with 10% calf serum (Fetalclone HI, Hyclone, Logan, UT). Infection of cells with SH/mR2 or control virus LXSH in the presence of polybrene was carried out [Miller et al, 1993], and stable infectants (>1 x 104 Printed from Mimosa WO 98/05769 -19- PCT/CA97/00540 clones) were obtained with hygromycin selection and pooled [Fan et al, 1996b; Miller et al, 1993]. Determinations of cell division times, plating efficiencies, and relative sensitivities to hydroxyurea cytotoxicity by estimating relative colony forming efficiencies, were earned out as previously desenbed [Lewis et al, 1978; Egan et al, 1987a, Hards and Wright, 1981]. 5 Growth in soft agar was estimated in 10 cm tissue culture plates containing 15 ml base agar (0 5% Bacto-agar in a-MEM plus 10% calf serum) and 10 ml of growth agar (0 33% agar in a-MEM containing 10% calf serum) Cells were obtamed from subconfluent cultures, and colonies were scored 10-15 days later [Egan et al., 1987a, 1987b, Hards and Wright, 1981] Transformation was also analyzed by determining focus formation after cells were infected 10 with SH/mR2 or LXSH or transfected with T-24 Ras or V12 Rac-1 plasmids by calcium phosphate precipitation [Taylor et al, 1992] At 40 hours after infection or transfection, cells were split into three 10 cm tissue culture plates which were provided daily with 20 ml of fresh complete medium (a-MEM plus 10% calf serum) for 10-14 days, stained with methylene blue and foci were scored [Taylor et al, 1992] The transfection frequency in all the expenments were 15 routinely determined by cotransfection of a mammalian expression plasmid for (3-galactosidase from Eshertchia colt, with the T-24 Ras or V-12 Rac-1 plasmids, followed by treatment of cells with the X-gal and counting the number of blue cells [Pnce et al, 1987]. In some cases, T-24 Ras plasmid transfected plates were selected with geneticin, and drug resistant colonies were scored approximately 14 later after staining with methylene blue 20 Assays for Tumorigemcity and Metastasis: Malignant potential was determined as desenbed herein above. Protein R2 Analysts: The procedures for Western blot analysis have been described previously, for example, using either the anti-myc mouse monoclonal 9E10 antibody (ATCC, Rockville, MD)[Fan et al, 1996b] or the anti-R2 rabbit polyclonal antibody [Chan et al., 1993] To 25 determine recombinant R2 protein expression during the cell cycle, flow cytometry analysis was performed following 9E10/fluorescein isothiocyanate antibody labelling as previously described [Blosmanis et al, 1987; Chadee et al, 1995]. Determination of Membrane-associated Raf-1 Protein: The membrane fraction was prepared as described by Qui et al. [1995], and used for Western analysis with a polyclonal antibody 30 specific for Raf-1 protein (Santa Cruz Biotechnology Inc, Santa Cruz, CA), after the protein content was determined by the standard Bio-Rad assay Densitometry analysis of the Raf-1 band was performed, and the amount of Raf-1 protein from each sample was corrected by densitometry analysis of a well separated band on a parallel gel stained with Coomassie blue Ribonucleotide Reductase Assay: The Assay was performed as described herein above In some 35 experiments enzyme assays were performed by combining purified recombinant Rl protein [Salem et al, 1993] with 9E10 antibody-precipitated R2 protein [Hurta and Wright, 1992] In this Example, 20 |i.g of the 9E10 antibody and 50 pi of Staphylococcal protein A-agarose (Sigma Chem Co., St Louis, MO) were added to 1 ml of the supernatant of centrifuged lysed Printed from Mimosa WO 98/05769 -20- PCT/CA97/00540 cells, and placed on a rocker at 4°C for 2 hours. The Staphylococcal protein A agarose-immunocomplex was washed three times with 1 ml of cold phosphate buffer containing 1 mg/ml bovine serum albumin The immunocomplex was then assayed for ribonucleotide reductase activity [Lewis et al, 1978; Hurta and Wright, 1992; Fan et al, 1996b, 5 Choy et al, 1988] Assay of MAPK Activity: Cultures with >90% confluency were stressed m serum-free medium [Stokoe et al., 1994; Jelinek et al, 1994] and extracted as previously described [Alessi et al, 1995]. MAPK-2 protein was immunoprecipitated by agarose beads conjugated with non-neutralizing antibody for the protein (Santa Cruz Biotechnology, Inc), and the kinase 10 activity of the immunocomplex was assayed by measuring its ability to phosphor/late myelin basic protein using a MAPK assay kit from Upstate Biotechnology, Inc (Lake Placid, NY) RESULTS Expression of Biologically Active R2 Protein. To determine the malignant potential of deregulated expression of the rate-limiting R2 component of ribonucleotide reductase, the 15 properties of cells stably infected with a retroviral expression vector (SH/mR2) carrying the R2 component [Fan et al., 1996b], were mvestigated The use of this expression vector allowed high infection efficiency and stable expression of the R2 protein To distinguish the vector gene product from the endogenous R2, a human c-Myc epitope coding for 10 ammo acids plus methionine was added to the 5'-end of the R2 cDNA Figure 1A shows that Western blots with 20 the 9E10 antibody that specifically recognizes the Myc-epitope sequence detects the R2 protein of approximately 45 kDa in SH/mR2 stably infected BALB/c 3T3 and NIH 3T3 cells (named B3/mR2 and N3/mR2, respectively), but not in control vector (LXSH) infected B3/SH or N3/SH cells. R2 specific antibodies detected the endogenous as well as the recombinant R2 protein in expression vector infected cells, and as expected only the endogenous protein was 25 observed in control vector infected cells (Fig IB) Flow cytometry analysis following 9E10/fluorescein isothiocyanate antibody labelling demonstrated that the recombinant R2 protein was constitutivelv expressed throughout the cell cycle (Fig 1C). Indirect microscopic analysis using the 9E10 antibody mdicated that essentially every cell in the B3/mR2 and N3/mR2 populations expressed the 30 Myc-tagged R2 protein. Several experiments were performed to demonstrate that the vector-expressed R2 is biologically active First, B3/mR2 and N3/mR2 cells were observed to be resistant in colony-forming experiments to the cytotoxic effects of hydroxyurea, an inhibitor of the R2 protein [Wright, 1989; Wnght et al, 1989], when compared to B3/SH and N3/SH cells [Fan et 35 al, 1996b] Second, ribonucleotide reductase activity was assayed and found that the CDP reductase activities in B3/mR2 and N3/mR2 cells m three independent experiments were 1.96 ± 0.32 and 1.71 ± 0.11 nmoles/mg protein/hour, respectively, which was 2 6 and 2 1 times higher than observed with B3/SH and N3/SH cells (0.74 ± 0.14 and 0 83 ± 0 08 nmoles/mg/hour, Printed from Mimosa WO 98/05769 -21- PCT/CA97/00540 respectively) Finally, enzyme assays were carried out by combining purified recombinant Rl protem [Salem et al, 1993], with 9E10 antibody precipitated R2 protein Significant levels of activity (15 to 20 nmoles/mg/hr) were detected when B3/n\R2 and N3/mR2 cells were used as a source for Myc-tagged R2, and as expected no activity was found when B3/SH or N3/SH cells 5 were used. Ras Transformation Potential Determined by Aberrant R2 Gene Expression. The above results indicate that cells can be altered in the regulation of biologically active R2 protem Therefore, altered R2 expression was tested to see if it further transformed cells like BALB/c 3T3 or NIH 3T3 Similar to control B3/SH and N3/SH cells, as well as the parental 10 non-infected lines, B3/mR2 and N3/mR2 cultures remained in a flat, non-transformed morphology on tissue culture plates, and exhibited contact and density inhibited growth (data not shown) No transformed foci were observed with BALB/c 3T3 or NIH 3T3 cells after infection with the retroviral SH/mR2 vector (Fig 2A, a and b) The results suggest that deregulation of R2 gene expression does not on its own 15 transform BALB/c 3T3 or NIH 3T3 fibroblasts To test the hypothesis that deregulated R2 expression may cooperate with oncogenes line H-ras, an expression plasmid containing T24 H-r«s was transfected into established recombinant R2 expressing cell populations derived from BALB/c 3T3 or NIH 3T3. A consistent and significant increase (3.4 fold) in the number of foci formed with H-ras transfected N3/mR2 cells was observed when compared to N3/SH 20 control cells (Fig. 2B, c and d and Fig. 2C). An even more marked increase of about 70 fold was observed when H-ras transfected B3/mR2 cells were compared to B3/SH cells (Fig 4B, a and b and Fig 2C). This occurred even though the transfection efficiency with NF3/mR2 and B3/mR2 cells as determined by scoring G418 selected colonies, and/or counting blue cells following cotransfection of H-ras with an expression plasmid for E coh _-galactosidase [Price et al, 25 1987], were actually lower (by about 50%) than with N3/SH and B3/SH cells Ras Malignancy Potential Determined by Aberrant R2 Gene Expression. Since combinations of altered R2 gene expression and activated H-ras were synergistic in focus forming experiments in which ras was transfected into altered R2 expressing cells, this gene combination was tested further by infecting four independent H-ras transformed 10Tl/2 cell 30 lines, CI, NR4, r-2 and r-3 that were previously characterized [Egan et al, 1987a, 1987b, Taylor et al., 1992; Stokoe et al., 1994], with the retroviral vector SH/mR2. Stable mfectants were selected with hygromycin, and Western blot analyses and enzyme activity assays confirmed that these infectants expressed biologically active Myc-tagged R2 protein Soft agar growth experiments revealed that H-ras transformed cells containing the 35 recombinant R2 sequence were much more efficient at producing colonies in semi-solid growth agar than the uninfected parental populations (e.g r-3) or control vector infected cells (CI, NR4, r-2) (Table 1). In addition, many of the colonies formed by cells infected with recombinant R2 were larger m size (Fig. 3A). Since each pair of recombinant R2 expressing and Printed from Mimosa WO 98/05769 -22- PCT/CA97/00540 control cell populations have almost identical growth rates (12.9 hours for Cl/SH and 12 2 hours for Cl/mR2,13.5 hours for r-2/SH and 13 9 hours for r-2/mR2,11 6 hours for r-3 and 11 9 hours for r-3/mR2,14.1 hours for NR4/SH and 14.3 hours for NR4/mR2), plating efficiencies (58% for Cl/SH and 55% for Cl/mR2,59% for r-2/SH and 63% for r-2/mR2,91% for r-3 and 88% 5 for r-3/mR2, 73% for NR4/SH and 75% for NR4/mR2), and cell cycle phase distributions (data not shown) when grown on solid surfaces, the alterations observed in soft agar and in foci forming experiments suggest that a combination of deregulated R2 expression and activated H-ras may lead to greater malignant potential m vivo Therefore, the tumongenic and metastatic potential of Cl/mR2 and Cl/SH cells was 10 compared in syngeneic C3H/HeN mice Marked differences in malignant potential were observed Cl/mR2 cells exhibited shorter tumor latency and greater tumor growth when compared to Cl/SH cells (Fig. 3B). Furthermore, metastasis assays clearly indicated that Cl/mR2 cells were more malignant than Cl/SH cells and produced significantly more lung tumors (Fig 3C). 15 R2 Gene Expression and Oncogene Cooperativity. The above results indicate that altered R2 expression can cooperate with activated H-ras in in vttro transformation and m m vivo malignancy assays Since no obvious differences in growth rates or cell cycle phase distributions were found that may account for this cooperation, as for example changes in cell cycle regulation, the following idea was tested Does deregulated R2 expression synergize 20 with ras by elevating the activity of a Ras signal pathway7 This would be consistent with studies showing a direct correlation between ras expression and malignant potential [Egan et al, 1987a, 1987b; Wnght et al, 1993, Bradley et al, 1986] A major Ras pathway for regulating gene expression involves the Raf-1 protein kinase. Activated Ras recruits Raf to the plasma membrane where Raf and downstream signalling molecules like MAPKs become activated 25 [Stokoe et al, 1994, Jelinek et al, 1994, Leevers et al, 1994] Usmg a Raf-1 specific antibody, the levels of membrane associated Raf-1 m six BALB/c 3T3, NIH 3T3 and 10T 1/2 derived cell lines containing deregulated R2 expression was compared with control cells containing only endogenous R2 protein (Fig. 4A) In all six cases, cell lines containing deregulated R2 showed increased membrane associated Raf-1, with an 30 average increase of about 30% which was highly significant (p < 0.001). In agreement with the above observation, cell lines with deregulated R2 expression exhibited a consistent and significant increase of about 70% (p < 0.001) in MAPK-2 activity (Fig. 4B) Oncogenic Ras also activates the Rac pathway which is parallel to the Raf pathway, and therefore constitutively active Rac-1 cooperates with membrane-targeted Raf-1 in malignant 35 transformation [Qiu et al, 1995] If MAPK activation mediated by Raf-1 translocation and activation is important m the R2/ras synergism desenbed herein above in this Example, then aberrant R2 expression should cooperate with activated Rac-1 in cellular transformation, because it has been shown Printed from Mimosa WO 98/05769 -23- PCT/CA97/00540 previously that activated Raf-1 and Rac-1 cooperate in mechanisms of transformation [Qiu et al, 1995). Figure 4C shows that this prediction is correct, since positive cooperation in transformation between activated Rac-1 and R2 was observed in a manner similar to Ras and R2, as measured by focus formation with N3/mR2 and N3/SH cells transfected with activated 5 V12 Rac-1 [Qiu et al, 1995] These observations are consistent with the view that deregulated R2 gene expression cooperates with oncogenes like ras and rac by upregulabng Raf translocation and MAPK pathway activity, but they do not rule out the possibility that other transduction pathways involving activated Raf may also be involved, since there is evidence that Raf can regulate some cellular activities through MAPK-independent pathway(s) [Lenormand et al, 10 1996, Koong et al, 1994, Agaiwal et al, 1995] This Example indicates for the first time that the R2 component of mammalian ribonucleotide reductase is a novel malignancy determinant that can synergize with activated oncogenes to modify malignant potential It is important to note that the only role ascribed to R2 m the cell prior to this Example is as a rate-limiting component of ribonucleotide reductase 15 This Example demonstrates that R2 can also participate in other critical cellular functions and can play a direct role in determining malignant potential through oncogenic cooperativity. EXAMPLE 2 R2 GENE EXPRESSION AND CHANGES IN DRUG SENSITIVITY AND GENOME STABILITY 20 MATERIALS AND METHODS Cell Ltnes and Culture Conditions: The hydroxyurea resistant mouse cell lines, H-2, H-4, LHF and SC2 were derived from mouse L cells and have been characterized in Choy et al [1988] and McClarty et al {1986] BALB/c 3T3 cells were used as recipients of an R2 retroviral expression vector (B3/mR2 and B3/R2c2 cell lines), or of the same retroviral vector lacking the R2 25 sequence (B3/SH cells)[Fan et al, 1996a; 1996b] NIH-3T3 cells were also used as recipients of the R2 retroviral expression vector (N/R2-4 cell line) or of this retroviral vector lacking the R2 sequence (N/SH cells), as desenbed previously [Fan et al, 1996a; 1996b] The N/R2+ASR2 cell line was the recipient through co-transfection using LipofectAmxne (Life Technologies, N.Y) [Damen et al., 1991] of retroviral vectors containing the R2 codmg sequence and the R2 30 sequence in the antisense orientation RP3 and RP6 cells are IOTV2 mouse cells that have been transfected with the T-24 H-ras oncogene and a mutant oncogenic form of the p53 gene (Taylor et al, 1992], and they were also used as recipients through transfection using LipofectAmine reagent, of a retroviral vector containing the R2 coding region in an antisense orientation [Fan et al, 1996b], to obtam RP3/ASR2 and RP6/ASR2 cells. IB cells are p53~/~ and were derived 35 from embryonic fibroblasts (Lowe et al.( 1994]. All cells were cultured in a-mimmal essential medium (Gibco, Grand Island, NY) containing 10% fetal bovine serum (Intergen, Purchase, NY) and antibiotics (100 umts/ml penicillin and 100 Hg/ml streptomycin) at 37°C m a humidified atmosphere containing 5% C02 Printed from Mimosa WO 98/05769 -24- PCT/CA97/00540 Drug Selections: Cells ranging in numbers from 500 to 1-2 x 105 were added to 100 mm tissue culture plates in growth medium containing 10% dialyzed fetal bovine serum, and in the absence or presence of drug [Huang et al., 1995a; Choy et al, 1988]. The culture medium was replaced with fresh medium every week for two to three weeks Surviving cells were 5 visualized by methylene blue staining, and colonies of about 50 cells or more were scored [Huang et al, 1995a] The relative colony forming efficiency was defined as the ability to produce colonies in the presence of a drug divided by that ability in the absence of drug. Assay for Gene Amplification: Genomic DNA was extracted from logarithmically growing cells by the phenol-chloroform extraction method [Blin and Stafford, 1976], and potential gene 10 amplification events were determined by Southern blot analysis as described [Huang et al, 1995a, Choy et al, 1988], using the cDNA fragments as probes noted below The pCAD142 plasmid containing CAD cDNA, which encodes the CAD protein complex [Shigesada et al, 1985], was used to obtain the 6.3 Kb Hind III fragment as a probe. The pLTR DHFR26 plasmid containing the mouse dihydrofolate reductase gene Chang et al, 1978], provided the 1 3 Kb 15 Bam HI fragment as a probe. The 1487 bp Sal 1/Pst I probe for ribonucleotide reductase R2 was prepared from cDNA clone 10 [Huang et al., 1995a; Choy et aL, 1988] Electrophoretic Gel Mobility Shift Assay (EMSA): EMSA was used to determine the presence of wild type p53 Assays were performed essentially as described [Price and Calderwood, 1993], with the following modifications Cells on 150 mm plates were washed once with ice 20 cold phosphate buffered saline (PBS) and scraped into 1 ml PBS. Cells were pelleted by centrifugation at 1300 g at 48C for 10 minutes and stored at -80°C Nuclei were prepared by lysing the pellets in 300 pi buffer A (20 mM HEPES |pH 7.6}, 20% glycerol, 10 mM NaCl, 1.5 mM MgCl2, 0 2 mM EDTA and 0.1% Triton X-100) for 20 minutes on ice Buffer A also contained 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM dithiothreitol (DTT) Nuclei were 25 isolated by centnfugation at 1300 g at 4°C for 10 minutes. Nuclear lysates were prepared by adding 20-40 |xl of buffer A containing 500 mM NaCl, 1 mM PMSF and 10 mM DTT to the nuclear pellet and incubating 20 minutes on ice The extracted nuclei were pelleted by centrifugabon at 16,000 g at 4°C; the supernatant was removed and an aliquot was used for protein determination using the Biorad protein assay procedure (Biorad) 30 The nuclear lysate was incubated with an excess of double stranded p53 consensus binding sequence (GGACATGCCCGGGCATGTCC)(SEQ ID No.162) end labeled with [Y-32P]-ATP using T4 polynucleotide kinase (Boehnnger). DNA binding was carried out in buffer containing 20 mM HEPES (pH 7 6), 20% glycerol, 1.5 mM MgCl2,0.2 mM EDTA, 1 mM PMSF and 10 mM DTT. Each binding reaction contained 5 (Ag cell lysate, 10 pg double stranded 35 poly (dI-dC)(Pharmacia), 1 4 ng labeled consensus probe and 100 ng of monoclonal antibody 421 (Santa Cruz) in a total volume of 20 |il DNA binding was allowed to proceed for 30 minutes at room temperature and the mixture was separated by electrophoresis on 5% nondenaturing polyacrylamide gels Electrophoresis was carried out at room temperature until the xylene Printed from Mimosa WO 98/05769 -25- PCT/CA97/00540 cyanol tracking dye had run to the bottom of the gel and the free probe had run off the gel Stattstocal Analysts-. Analysis of covariance was used to compare dose response data between groups of different cell lines, with the significance level set at a = 0 05 [Huang et al., 1995a] RESULTS 5 Hydroxyurea Resistant Cell Lines with Decreased Sensitivity to Non-selective Drugs H-2, H-4, LHF and SC2 are mouse L cell lines selected for resistance to the cytotoxic effects of the antitumor agent, hydroxyurea These four cell lines exhibited resistance to hydroxyurea in colony forming efficiency experiments, that ranged between approximately 18 (H-2) to 30 (SC2) fold higher than the wild type mouse L cell line from which they were 10 derived [Choy et al, 1998, McClarty et al., 1988] They also contained elevated levels of ribonucleotide reductase activity that ranged between 2 2 fold (H-2) to 17 fold (LHF and SC2), which was primarily due to increases in the R2 component of ribonucleotide reductase that is limiting for enzyme activity and cell division in proliferating mouse cells Table 2 shows that the four hydroxyurea resistant cell lines were also less sensitive to the cytotoxic effects of 15 N-(phosphonacetyl)-L-aspartate (PALA) and methotrexate (MTX) in colony forming experiments, when compared to parental wild type mouse L cells These differences in drug sensitivity are highly significant, with p values of <0 0001 for each of the cell lines when compared to the parental wild type mouse cells. Although many mechanisms responsible for drug resistance have been described 20 [Wfight, 1989; Kohn, 1996], resistance to MTX and PALA are frequently accompanied by increased levels of the drug targeted gene products, dihydrofolate reductase (DHFR) or CAD (a multifunctional polypeptide containing carbamyl phosphate synthetase, aspartate transcarbamylase and dihydrooratase) respectively, and this often occurs through a mechanism of gene amplification [Huang et al., 1995a, Livingston et al., 1992, Yin et al., 1992, 25 Mai, 1994, Stark, 1993] Indeed, the principal and perhaps only mechanism for PALA resistance in mouse cells occurs via CAD gene amplification [Stark, 1993] Therefore, colonies that developed in the presence of normally cytotoxic concentrations of these two drugs were examined for possible gene amplification events. Figure 5 shows that cells that proliferated in the presence of PALA or MTX exhibited increased CAD or DHFR gene copy numbers. In keeping 30 with previous studies [Stark, 1993; Huang et al., 1995b; Otto et al, 1989; Stark et al., 1990], all colonies that developed in PALA and tested (10/10) showed CAD gene amplification Also as previously reported [Huang et al., 1995b], some but not all colonies that developed in the presence of MTX (3/6) showed DHFR gene amplification. Direct Test for a Relationship Between R2 Gene Expression and Decreased Drug 35 Sensitivity. Since hydroxyurea resistant mouse cells contain other biochemical alterations m addition to changes in ribonucleotide reductase [Wright et al, 1989], the relationship between drug sensitivity and increased R2 levels was directly tested with cells containing a retroviral expression vector encoding the mouse R2 sequence, and cells containing the same retroviral Printed from Mimosa WO 98/05769 -26- PCT/CA.97/00540 vector but lacking the R2 sequence B3/mR2 is a population of BALB/c 3T3 cells containing elevated R2 protein due to the presence of a retroviral expression vector encoding R2, and B3/SH is a cell population that has wildtype levels of R2 protein and contains the empty vector as a control. B3/R2c2 is a cloned line with elevated R2 protem selected from the 5 B3/mR2 population. Consistent with previous reports showing that elevations in R2 gene expression leads to resistance to hydroxyurea, Table 3 shows that B3/mR2 and B3/R2c2 cells are significantly more resistant to the cytotoxic effects of hydroxyurea, at a range of concentrations, when compared to B3/SH cells These results further demonstrate that B3/mR2 and B3/R2c2 cells 10 express increased levels of an active R2 component of ribonucleotide reductase B3/mR2 and B3/R2c2 cells were also significantly less sensitive to the cytotoxic effects of PALA and MTX, which act at sites other than ribonucleotide reductase (Table 3). Resistance to these two drugs ranged between approximately 10 fold with 100 nM MTX to more than 100 fold at most concentrations of PALA tested 15 Furthermore, Southern blot analysis showed that colonies that developed in the presence of PALA or MTX contained amplifications of CAD or DHFR genes (Figure 6), although as observed with mouse L cells (Figure 5) and as has been reported in other studies [Hurta and wright, 1992; Hurta et al, 1991], not all colonies that developed m MTX containing medium exhibited DHFR gene amplification Unlike PALA resistance, MTX resistance in mouse cells 20 can occur through a variety of mechanisms [Otto et al, 1989; Stark et al, 1990, Flintoff, 1989] The changes in sensitivity to chemotherapeutic compounds exhibited by cells containing elevated levels of the ribonucleotide reductase R2 component were fu-ther tested using NTH-3T3 cells containing the R2 expression retroviral vector (Table 4). These cells (N/R2-4) were resistant to hydroxyurea when compared to cells contammg the retroviral 25 vector lacking the R2 coding sequence (N/SH) The N/R2-4 cells were also significantly more resistant to MTX. Although the N/R2-4 cells showed a trend towards resistance to PALA when compared to N/SH cells, this trend was not statistically significant This latter observation indicates that other factors inherent in the genetic differences between the cell lines used in this study, in addition to the increased R2 levels, can influence drug sensitivity 30 responses Therefore, the hypothesis that R2 levels are important in determining drug sensitivity characteristics was tested by investigating drug sensitivities after decreasing the levels of R2, through expression of an R2 antisense construct introduced into N/R2-4 cells to produce the N/R2+ASR2 population Figure 7 shows that the level of R2 protem is markedly 35 reduced m N/R2+ASR2 cells when compared to N/R2-4 cells The N/R2+ASR2 cells were significantly more sensitive to hydroxyurea, PALA and MTX when compared to N/R2-4 cells (Table 4) Furthermore, sensitivity to these three drugs in the R2 antisense expressing cells was significantly increased when compared to control N/SH cells contammg the empty vector Printed from Mimosa WO 98/05769 -27- PCT/CA97/00540 (Table 4) Mouse 10T^ cells transfected with activated ras and a mutant oncogenic form of p53 are highly resistant to chemotherapeutic agents [Huang et al, 1995b]. The observation that R2 antisense expression can increase sensitivity of N1H-3T3 cells to hydroxyurea, PALA and 5 MTX lead us to test the possibility that cells containing ras and mutated p53 may also exhibit reduced drug resistance characteristics in the presence of an R2 antisense sequence Table 5 shows that this is correct Cells containing the R2 antisense sequence are significantly more sensitive to hydroxyurea, PALA, and MTX when compared to cells containing the same vector but without R2 in the antisense orientation These observations suggest that at least one of the 10 determining factors relevant to drug sensitivity of these highly transformed and malignant cells, is ribonucleotide reductase R2 levels Evidence that Loss of p53 Protein Function is not Required for R2-Mediated Drug Resistance and Gene Amplification. Inactivation or loss of p53 is a common event associated with the development of tumors and the accompanying decrease in genetic stability observed 15 in malignant cells, including the ability to undergo spontaneous gene amplifications [Liningston et al, 1992; Yin et al, 1992; Takenaka et al., 1995] Therefore, we tested the possibility that the increased drug resistance properties exhibited by the R2 overproducing B3/mR2 and B3/R2c2 cells may be occurring through a mechanism that results m a loss of wild type p53 activity. It has been demonstrated that p53 is a transcription factor, and that transactivation 20 by wild type p53 but not mutated versions of p53 is sequence-specific, and correlates with its binding to consensus DNA sequences [Takenaka et al, 1995, Kern et al., 1992; Funk et al, 1992] To determine the presence or absence of wild type p53 function in drug resistant colonies that developed in the presence of PALA, MTX or hydroxyurea, cell extracts were used m electrophoretic gel mobility shift assays (EMSA) [Price and Calderwood, 1993], to test for 25 sequence specific p53 binding activity Figure 8 shows that drug resistant clones derived from R2 overexpressing cells exhibited wild type p53 binding activity. These observations also agreed with our inability to detect mutant p53 proteins in cells from drug resistant colonies in immunoprecipitation assays usmg the Pab240 monoclonal antibody [Gannon et al, 1990], which specifically detects common forms of mutant p53 30 EXAMPLE 3 ANTISENSE DEOXYRIBONUCLEOTIDE SEQUENCES THAT TARGET RIBONUCLEOTIDE REDUCTASE AND ARE CYTOTOXIC FOR HUMAN TUMOR CELLS. As shown m the Examples herein above full length antisense constructs of R2 affect the tumongenicity and/or metastatic competence of tumor cells and susceptibility to 35 chemotherapeutic agents Applicants therefore investigated the potential of shorter antisense constructs of Rl and R2 for their effect on tumor cells MATERIALS AND METHODS Colony Formtng Efficiency and Treatment of Cells -unth Antisense Constructs• Colony forming Printed from Mimosa WO 98/05769 -28- PCT/CA97/00540 efficiency was determined as previously reported [Huang and Wright, 1994] The cells were cultured for 24 hours at 37°C in growth medium with 10% fetal bovine serum The cells were washed in 5ml phosphate buffered saline, pH 7.2, once prior to hpofectin +/- oligonucleotide treatment. 5 The oligonucleotides being tested were added to cell cultures in the presence of 2.5 fig of DOTMA/DOPE (Lipofectin, Life Technologies, Inc.) for four hours The oligonucleotide was tested at 0.2 nM unless otherwise indicated. Controls were the cultures treated with lipofecbn but without the oligonucleotide. After 4 hours the medium containing the oligonucleotide was removed and washed with 5 ml of growth medium The cells were then cultured in growth 10 medium containing 10% fetal bovine serum for seven to ten days Surviving cells were visualized by methylene blue staining, and colonies were scored. In some experiments cell aliquotes were removed from the culture and viability was determined using the trypan blue exclusion test [Phillips, 1973] Results were analyzed as percent of surviving cells compared to control cells 15 RESULTS Antisense molecules were identified that target ribonucleotide reductase As shown below they were cytotoxic for a variety of human tumor cells Sequences were found that facilitated drug-cytotoxicity for drug resistant tumor cells. That is, at very low non-cytotoxic concentrations, antisense sequences targeting ribonucleotide reductase can sensitize tumor cells 20 to the cytotoxic activity of clinically important chemotherapeutic compounds In initial studies two antisense sequences of 20-mer, designated AS-II-336-20 and AS-II-2229B-20, directed against the R2 mRNA were made and investigated The first, AS-II-336-20, has the sequence 5-TCC TGG AAG ATC CTC CTC GC-3'(SEQ ID No:l), and targets the R2 message of human ribonucleotide reductase at nucleotides 336-355, based on the 25 numbering of R2 nucleotides [Pavloff et al., 1992]. The AS-H-2229B-20 sequence is 5-TCC CAC ATA TGA GAA AAC TC-3' (SEQ ID No:2), and targets the R2 message at nucleotides 2229-2248 Both AS-II-336-20 and AS-II-2229B-20 were constructed as phosphorothioate sequences to protect against nuclease activity [Anazodo et al., 1995] Antisense construct AS-II-336-20 was tested for the ability to inhibit the 30 proliferation of human tumor cells (Hela) in relative colony forming efficiency experiments as described herein above. Hela S3 cells (American Type Culture Collection, Rockville, Maryland, ATCC), and a Hela cell line (Hela ImM) previously selected for resistance to the antitumor agent, hydroxyurea [Wright et al, 1987], were tested (Table 6) Two experiments were undertaken with Hela S3 cells With a 4 hour treatment of 0.2 fiM antisense construct 35 AS-II-336-20, inhibition of 92% and 827o was seen in colony forming efficiency in two experiments, respectively The same experiment was repeated with the Hela ImM cell line and with varying concentrations of the antisense construct AS-II-336-20 (Table 6) with similar results, 0.2 |iM was an effective concentration for inhibiting colony formation Printed from Mimosa WO 98/05769 -29- PCT/CA97/00540 These data show that AS-II-336-20 is a very effective inhibitor of human tumor cell colony forming ability, and it is effective both in inhibiting the proliferation of human tumor cell colony forming ability and in inhibiting the proliferation of human tumor cells that exhibit resistance to another chemotherapeutic compound Similarly, as shown in Table 6, 5 antisense construct AS-II-336-20 is an effective antitumor compound in experiments performed with the mouse tumor cell line, SC2, which is a highly hydroxyurea resistant mouse L cell line [McClarty et al., 1988]. The antisense sequence AS-II-2229B-20 was also tested for the ability to inhibit the proliferation of human Hela tumor cells in relative colony forming efficiency experiments with 10 results similar to that of AS-II-336-20 as shown in Table 6 These data show that AS-II-2229B-20 is a potent antitumor agent when tested with Hela S3 cells and with the drug resistant Hela ImM cell line The antisense construct AS-II-2229B-20 was also tested for the ability to inhibit the proliferation of the human breast cancer cell line MDA435 and found to be very effective (Table 8) 15 The ribonucleotide reductase R2 antisense construct designated AS-II-2229B-20 was tested for tumor cell cytotoxicity by comparing the results obtained with human tumor and non-tumor cell populations Hela S3 tumor cells and WI 38 normal non-tumorigenic human cells were used. Tumor cells were found to be much more sensitive to the cytotoxic effects of AS-II-2229B-20 than normal non-tumorigenic cells For example, analysis of cells three days 20 after antisense exposure indicated that tumor cells were approximately 5-times more sensitive to the cytotoxic effects of AS-II-2229B-20 than normal non-tumorigenic cells averaged over 4-8 determinations These results indicate that short oligodeoxyribonucleotide sequences in an antisense orientation are excellent antitumor agents, and suggest that other antisense constructs that 25 target the R2 message may have similar properties The best antitumor agents would be those that exhibit suitable energy related characteristics important for oligonucleotide duplex formation with their complementary templates, and which show a low potential for self-dimerization or self-complementation [Anazodo et al., 1996] An analysis of the R2 mRNA using a computer program (OLJGO, Primer Analysis Software, Version 3.4), was earned 30 out to determine antisense sequence melting temperature, free energy properties, and to estimate potential self-dimer formation and self-complimentary properties [Anazodo, et al., 1996], of a series of additional antisense sequences (Table 7, SEQ. ID NOS 3-102) designed to target the R2 message. Table 7 shows a list of the additional R2 antisense inhibitors, with appropriate properties. 35 To test the antisense effects of many of these sequences, as phosphorthioate deoxyribonucleobdes, they were examined in relative colony forming experiments performed with a series of human tumor cell lines Many of these antisense constructs, as predicted, are potent inhibitors of human tumor cell proliferation. For results obtained with cancer cells Printed from Mimosa WO 98/05769 -30- PCT/CA97/00540 derived from the bladder, breast, lung, colon, pancreas, prostate, liver and cervix, see Table 12 In addition, m vivo studies with AS-II-626-20 were undertaken m C3H/HeN mice as reported in Table 13 and show a significant reduction in metastasis in the antisense treated mice. Based on Example 2, treatment of human tumor cells with very low concentrations of 5 short antisense sequences was tested to determine if these constructs could sensitize the tumor cells to inhibitory effects of other chemotherapeutic drugs The concentration used was not cytotoxic in itself as shown in Table 6 The treatment of Hela S3 and Hela ImM cells with 0 02 |J.M of the AS-II-2229B-20 antisense construct increases the sensitivity of these cells to N-(phosphonacetyl)-L-aspartate (PALA) and to methotrexate (MTX) as shown in Table 9 10 These observations indicate that antisense compounds targeting the R2 message can act synergistically with well known chemotherapeutic agents. Ribonucleotide reductase is composed of two dissimilar protein components coded by two distinct genes, Rl and R2. Therefore, the results desenbed hereinabove suggest that the Rl message may also be an appropriate target for designing short antisense molecules that have 15 potent antitumor activity To test this possibility a 20-mer deoxyribonucleotide phosphorothioate sequence m antisense orientation, designated AS-I-1395-20, was constructed and its antitumor abilities were tested. The antisense construct AS-I-1395-20 has the sequence 5'-ACA GGA ATC TTT GTA GAG CA-3' (SEQ ID No:103), and targets the Rl -message at nucleotides 1395-1414 As shown in Table 10 it is an effective inhibitor of tumor cell 20 proliferation using Hela S3 cells and Hela ImM drug resistant cells These results demonstrate the usefulness of designing antisense sequences that target the Rl message, and suggest that other potential sites may also be effective Therefore, the Rl mRNA was analyzed in a search for antisense oligodeoxynbonucleotide sequences that exhibit suitable characteristics (as done for R2 mRNA and described above). Table 11 provides a list of additional antisense sequences 25 with characteristics that are consistent with being antitumor agents EXAMPLE 4 INHIBITION OF TRANSFORMATION BY R2 ANTISENSE Utilizing the methods set forth in Examples 1-3, the inhibition of transformation of mammalian cells by treatment with the R2 antisense sequence of the R2 coding region [Fan et 30 al, 1996b] was undertaken. NIH-3T3 mouse cells containing the H-ras oncogene were transfected with either the antisense orientation of the R2 coding sequence or the sense orientation of the R2 coding sequence. The results shown in Fig. 9 demonstrate that m the presence of the R2 antisense construct there was a decrease in transformed foci and reduced soft agar growth (Fig 9, lane b) compared to the control cells (Fig. 9, lane a). As shown in Example 1, herein above, 35 the R2 coding region can cooperate with H-ros to enhance malignancy as shown by the increased number of transformed foci (Fig 9,lane c) Furthermore, colony efficiency assays performed in soft agar as described herein demonstrated similar results Colony forming efficiencies of 15.6±6 73 for NIH-3T3 mouse cells Printed from Mimosa WO 98/05769 -31- PCT/CA97/00540 containing the H-ras oncogene, 4 4+2 62 for NIH-3T3 mouse cells containing the H-ras oncogene and the R2 antisense sequence, and 51±12 29 for NIH-3T3 mouse cells contammg the H-rfls oncogene and the coding region sequence for R2 were seen. EXAMPLES 5 Western blot analysis of AS-II-626-20 inhibition of ribonucleotide reductasp R2 protein level in L60 mouse tumor cells. Cells were treated for 4 hours with growth medium supplemented with hp of ec tin but without antisense oligonucleotides (a) or with hpofectin medium containing 0 2 |±M AS-II-626-20 (b) As added controls the tumor cells were also treated for 4 hours with growth medium supplemented with lipofectin and 0.2 (iM oligonucleotide scrambled control, 10 which contains the same proportion of nucleotides found in AS-II-626-20 but in a different order (ACGCACTCAGCTAGTGACAC, SEQ ID. NO 164) (c) or with 02 (iM mismatch oligonucleotide, which contains a four nucleotide mismatch mutation when compared to AS-II-626-20 (TCGC changed to CTGC) (d) Note the significant decrease in R2 protein in tumor cells treated with AS-II-626-20 (b) when compared to the controls (a, c and d) 15 Decrease in R2 protein levels in mouse L60 tumor cells following treatment with a variety of R2 antisense oligonucleotides, as determined by Western blot analysis. Cells were treated for 4 hours with 0.2 |iM oligonucleotide in the presence of lipofectin (b to f), or with hpofectin without oligonucleotide as a control (a) (b) Cells treated as AS-II-667-20, (c) cells treated with AS-II-816-20; (d) cells treated with AS-II-1288-20; (e) cells treated with AS-II-20 1335-20 and, (f) cells treated with AS-II-1338-20. Note the decrease in R2 protein levels in cells treated with antisense oligonucleotides that target the R2 mRNA, in keeping with their abilities to inhibit human tumor cell proliferation (Table 12). Throughout this application, various publications, including United States patents and published patent applications are referenced by author and year or number Full citations 25 for the publications are listed below The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of 30 description rather than of limitation. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described. Printed from Mimosa WO 98/05769 PCT/CA97/00540 -32- TABLE1 INCREASED COLONY FORMATION IN SOFT AGAR BY ras-TRANSFORMED CELLS CONTAINING THE RECOMBINANT R2 VECTOR Cell Line Colonies (average ± SE) formed m soft agar with varying cell inoculum3 10 1(P 10* 105 Cl/SH 0 4±3 66±9 Cl/mR2 3±3 28±7 347 1 45 15 r-2/SH ND 9±2 105 ±7 r-2/mR2 ND 24 ± 1 298 ±11 NR4/SH 0 311 32 ±4 NR4/mR2 2± 1 14 ±2 127110 r-3 7± 1 100 ±11 ND 20 r-3/mR2 31 ±4 309 ±17 ND a The number of colonies presented were the results obtained in three independent experiments, except those obtained for r-2/SH and r-2/mR2 cells which were the results from single 25 experiments with triplicate dishes ND, not determined. Printed from Mimosa WO 98/05769 PCT/CA97/00540 -33-TABLE2 DRUG SENSITIVITIES DETERMINED BY RELATIVE COLONY FORMING EFFICIENCIES xl(H A. PALA DRUG CELL LINES CONC. W.T. H2 H4 LHF SC2 20 nM 30 nM 40jiM 50 |iM 60 |iM 172 3+126.3 50 3 ± 20 5 15 0± 7 0 3.6 ±1.1 1 3± 04 406 7 ± 202 2 39.4 ± 16.4 23 3 ± 10 4 7 9 ± 1 7 3.6 + 0 6 322.5 ±36 4 84 0 ± 30 0 43 3 ± 9.6 23 2 ± 0.5 11 1± 14 233 3 + 3 6 78 8 ± 7 9 46 5 ±9.9 25.0 ±6.8 10 7±30 850 1 ± 325 2 187 6 ± 46 4 37.5 ± 8.7 47.5 ±35 8 176+12 B. MTX CONC. W.T. H2 H4 LHF SC2 40 nM 60 nM 80 nM 100 nM 150 nM 11 2±72 12 3 ± 7 2 2.2 ± 1 6 0.8 ± 0.4 0.5 ± 0.2 52 6 ± 25 2 73 7 ± 16 6 67 7 ± 20 0 75 3 ± 100 53 3 ± 9 4 44 2 ± 20 9 347+112 39.3 ±18 7 15 1 ± 8.8 32.3 ± 13 7 143.4 ±41.3 63 5 ± 18.6 68 2 ± 19.2 60 8 ±16.7 63 9 ± 16 0 880 4 ±147 4 566 8 ± 66.2 306.6 ± 61 5 261 8 ± 39.7 301.6 ±76.8 35 The relative colony forming efficiencies are shown ± se, and the values presented are from 4 to 8 determinations Statistically significant differences were observed when data obtained with H2 (p = 0.0004), H 4 (p £ = 0 0001), LHF (pSO 0001), and SC2 (p < 0 0001) were each compared to data obtained with the parental wild type (W.T.) cell line. Printed from Mimosa WO 98/05769 PCT/CA97/00540 -34- TABLE3 DRUG SENSITIVITIES DETERMINED BY RELATIVE COLONY FORMING EFFICIENCIES xl(H A. HYDROXYUREA 10 DRUG CELL LINES CONC B3/SH B3/mR2 B3/R2c2 0.1 mM 0.4 mM 0.5 mM 0.6 mM 0.8 mM 33 ±14 0.17 ±0.19 0 21 ±014 0 41 ± 0.22 0 19 ±0 62 1310 ±319.0 14.6 ±40 65±46 52137 26±1 4 830 8 1 97.0 33 71110 26,9111 9 1251 4.6 13 21 6.4 B PALA CONC B3/SH B3/mR2 B3/R2c2 10 |iM 20 |JM 40 MM 50 nM 60 pM 80 nM 17.91110 0 39 10.18 0 3510.01 0 24 ± 0 14 0.12 ±0.05 0.17 ±0.08 9650 ±529.7 1201 ± 28 4 25.0 ±4.6 27.6 ±89 250 ±64 27.116.75 1230.0 1 97.0 55.1115.6 20 2 1 6 8 15.914 0 18 715.3 20.014.9 C. MTX CONC B3/SH B3/mR2 B3/R2c2 20 nM 192 6 1 44.6 1055 0 1 239.0 382 4 1 71 3 40 nM 15.712.9 62118 8 60.8113 0 60 nM 61120 76 7 1 216 64.1120.5 80 nM 2.2 1 0.7 17.513.6 20.11 55 100 nM 15105 123128 21.01 72 150 nM 3011.1 23.017.6 33.4114.3 45 The relative colony forming efficiencies are shown ±SE, and the values presented are from 4 to 12 determinations. Statistically significant differences were observed when data obtained with B3/mR2 or with B3/R2c2 were compared with data obtained with B3/SH (all p values 50 were £ 0.0001 for data obtained in the presence of hydroxyurea, PALA or MTX) Printed from Mimosa WO 98/05769 PCT/CA97/00540 -35- TABLE 4 DRUG SENSITIVITIES DETERMINED BY 5 RELATIVE COLONY FORMING EFFICIENCIES xltH A.HYDROXYUREA DRUG CELL LINES 10 CONC N/SH N/R2-4 N/R2+ASR2 03mM 1.14±012 461 ±98 0.49 ±0 34 04 mM 0 71 ±0.17 18.0±67 014±014 B. PALA CONC N/SH N/R2-4 N/R2+ASR2 10 nM 5 28 ± 1 5 6 22 ± 3.3 1.81 ±08 15 nM 5 83 ± 2 7 10.0 ±55 0.58 ± 0.3 20 MM 0 30 ± 0 1 1.71 ± 1 2 0 04 ± 0 04 25 nM 0.53 ± 0.3 08 ±0.7 0 04 ± 0 04 30 MM 0.48 ± 0.08 103 + 007 012 ±0.12 40 nM 0.27 ±0.2 0.14 ±0 08 0 04 ±0.04 C. MTX CONC N/SH N/R2-4 N/R2+ASR2 20nM 655± 74 8 540 ± 25 1 423 + 119 35 40nM 21 ± 121 147± 4.2 35±1.9 60nM 3 4 ± 2 2 62.2 ± 30 7 1 9 ± 1 3 80nM 5 0± 5 0 504±239 25±15 lOOnM 42± 25 66.1±32.8 11±06 150nM 14± 0.9 21.0±115 0,n=4 The relative colony forming effeciencies are shown ± SE, and the values presented are from 4 to 6 determinations. Where 0 is shown the number of determinations using 1 x 105 cells per test is shown as 4 (n=4). Statistically significant differences were observed when data obtained with N/SH in the presence of PALA was compared to data obtained with N/R2-4 or with 45 N/R2+ASR2 in the presence of hydroxyurea (p = 0.0001 in both cases) or in the presence of MTX (p = 0 0002 and 0.032, respectively) Statistically significant differences were also observed when data obtained with N/SH in the presence of PALA was compared to data obtained with N/R2+ASR2 (p = 0.002), but not with data obtained with N/R2-4 Printed from Mimosa 10 WO 98/05769 PCT/CA97/00540 -36- TABLE 5 DRUG SENSITIVITIES DETERMINED BY RELATIVE COLONY FORMING EFFECIENCIES xlCH A. HYDROXYUREA DRUG CONC CELLUNES RP3/SH RP3/ASR2 RP6/SH RP6/ASR2 15 01 mM 0 2mM 0 3mM 0 4mM 0 5mM 0 6mM 0 8mM 263 6 ± 19 3 5361137 20 8 ± 7 5 5 8 ± 1 9 4 8 ± 1 9 07 + 03 08 + 03 109.8 ±43 22 9 ± 3 1 6.6 ±25 10 + 02 0 2 ± 0 1 0 3 ± 0 1 0 110 05 201 3 ± 27.2 35 5 ± 8 4 12.6 ±24 10 8 ± 4 1 12.1 ±39 6 6 ± 2 9 1 7± 1 2 43 8 ± 12 3 86 ±2.5 4 5 ± 1 1 1.2 ± 0 5 1 8 ±0 9 1.5 ±0 7 04 ±0 3 B. PALA CONC RP3/SH RP3/ASR2 RP6/SH RP6/ASR2 10nM 20|iM 30jiM 40|iM 30 50|iM 60nM 2569 ±338 123 4 ±193 45.2 ±78 15.0 ±4 9 9 3 ± 3 6 3.9 ± 1 6 1183 ±384 86 1 ± 32 9 195 ±4 7 4 7 ±0 6 21 ±08 0 3 ± 0 2 4619 ±648 1220 ±255 4501129 271 ± 68 109 ± 23 55.5 ± 13 2083 ±960 368 ±154 316 ±171 116± 54 41.7 ± 23 13 2 ± 6.3 C. MTX CONC RP3/SH RP3/ASR2 RP6/SH RP6/ASR2 20nM 961.7 ±134 485 9 ±165 1856 ±464 1504 1486 40nM 347.1 ± 154 77 8 ±18 172 ± 413 91.5 ± 28 1 60nM 123 8 ± 64 18 1 ± 6.2 77.3 ± 15 6 49 9 ± 14.1 80nM 66.5 ± 37 4.4 ±0.8 68.7 ± 16 7 36.016.0 lOOnM 34.8 ± 21 0 6 ± 0.06 46.6 ± 5.6 14.41 3.8 150nM 4 7±3 0 2 ± 0.1 11.1 ± 4.4 3.5 ±0.9 The relative colony forming effeciencies are shown ± SE, and the values presented are from 4 to 10 determinations. Statistically significant differences were observed when data obtained with RP6/SH was compared with data obtained with RP6/ASR2 (p = 0.0001,0.0001 and 0.0001 in the presence of hydroxyurea, PALA and MTX, respectively). Significant differences were 50 also observed when data obtained with RP3/SH was compared with data obtained with RP3/ASR2 (p = 0 04, 0.0001 and 0.004 in the presence of hydroxyurea, PALA and MTX, respectively). Printed from Mimosa WO 98/05769 PCT/CA97/00540 -37- TABLE6 REDUCED COLONY FORMING EFFICIENCY FOLLOWING TREATMENT WITH R2 ANTISENSE CONSTRUCTS CELL LINE Hela S3 10 Cone % Inhib Cone % Inhib AS-II-336-203 AS-n-2229B-20b 15 0.2 (iM 92% 005nM 50% 0.2 pM 82% 0.10 >iM 0.20 n M 0 20 nM 80% 95% 97% 20 CELL LINE Hela ImM Cone % Inhib. Coric % Inhib. AS-II-336-20a AS-II-2229B-20b 25 0 0 001 JIM 15% 001 nM 0% 0.05 pM 25% 002 nM 0% 30 OlOuM 60% 0 03 piM 21% 0.20 (iM 85% 0.04 005 HM 005 fiM 0.10 jiM 34% 48% 50% 78% 35 CELL LINE. Mouse SC2 0.20 pM 0.20 nM 97% 90% 40 Cone AS-II-336-20a % Inhib. 0 02|lM 95% Printed from Mimosa WO 98/05769 -38- PCT/CA97/00540 TABLE 7 ANTISENSE SEQUENCES DESIGNED TO TARGET THE R2 MESSAGE SEQ ID No: Name Sequence 5-3' Tm TC dG kDa/mol SEQ ID No:3 AS-II-6-20 ACCCTTCCCATTGGCTGCGC 62 8 -45 5 SEQ ID No:4 AS-II-13-20 GsCCsTCCGs ACCsC 1' 1 CsCCs ATI sG 60 1 -43 7 SEQ ID No:5 AS-II-14-20 TGCCTCCGACCCTTCCCATT 60 1 -43 7 SEQ ID No:6 AS-II-16-18 TGCCTCCGACCCTTCCCA 58 4 -40 3 SEQ ID No:7 AS-I1-75-20 CsGCGsCGCsTCCsCGGsCCCsTTCsC 72 7 -53 7 SEQ ID No:8 AS-II-75-20 CGCGCGCTCCCGGCCCTTCC 72 7 -53 7 SEQ ID No:9 AS-II-79-14 CGCGCTCCCGGCCC 59 1 -38 8 SEQ ID No:10 AS-II-109-20 CsCCCsTCACsTCCsAGCsAGCsCTsT 57 9 -41 8 SEQ ID No:ll AS-II-110-20 ACCCCTCACTCCAGCAGCCT 57 3 -41 2 SEQ ID No:12 AS-II-114-20 GGCGACCCCTCACTCCAGCA 61 8 -43 2 SEQ ID No:13 AS-II-127-12 GCACGGGCGACC 41 7 -28 8 SEQ ID No: 14 AS-II-130-20 TGGGACAGGGTGCACGGGCG 67.6 -46 7 SEQ ID No:lS AS-II-134-20 GACGGCTGGGACAGGGTGCA 62 6 -43 2 SEQ ID No:16 AS-II-151-20 GAGCAGCCAGGACAGGACGG 59 3 -41.7 SEQ ID No: 17 AS-II-163-20 GsCGsAAGsCAGsAGCsGAGsCAGCsC 62 1 -44 3 SEQ ID No:18 AS-II-166-20 GCAGCGAAGCAGAGCGAGCA 61 4 -43 1 SEQ ID No: 19 AS-II-185-20 GGGAGAGCATAGTGGAGGCG 56 0 -40 9 SEQ ID No:20 AS-II-189-20 CGGAGGGAGAGCATAGTGGA 54.1 -39.4 SEQ ID No:21 AS-n-201-20 GCGAGCGGGACACGGAGGGA 63 5 -45.1 SEQ ID No:22 AS-n-217-20 CGGGTCCGTGATGGGCGCGA 69 5 -48 8 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 -39- PCT/CA97/00540 SEQ ID No:23 AS-II-225-20 AGCTCCTGCGGGTCCGTGAT 61 4 SEQ ID No: 24 AS-II-253-14 CCCCTTCAGCGGCG 50 8 SEQ ID No:25 AS-II-280-20 CGGCGGCGTGTTCTCCTTGT 61 8 SEQ ID No:26 AS-II-288-12 CGGCGGCGTGTT 43 2 SEQ ID No:27 AS-II-323-20 TCCTCGCGGTCTTGCTGGCC 64 1 SEQ ID No;28 AS-II-344-20 CCGTGGGCTCCTGGAAGATC 58 0 SEQ ID No:29 AS-II-362-20 CTGCTTTAGTTTTCGGCTCC 51 2 SEQ ID No:30 AS-II-391-17 CGGCTCATCCTCCACGC 54.5 SEQ ID No:31 AS-n-404-20 GGTTTTCTCTCAGCAGCGGC 56 4 SEQ ID No:32 AS-II-412-20 GCGGCGGGGGTTTTCTCTCA 62 8 SEQ ID No:33 AS-II-414-20 AAGCGGCGGGGGTTTTCTCT 60 7 SEQ ID No:34 AS-II-425-20 GGAAGATGACAAAGCGGCGG 59 1 SEQ ID No:35 AS-II-439-20 ATGGTACTCGATGGGGAAGA 50 8 SEQ ID No:36 AS-II-472-20 AGCCTCTGCCTTCTTATACA 46 1 SEQ ID No:37 AS-II-494-20 CCTCCTCGGCGGTCCAAAAG 60 4 SEQ ID No:38 AS-I1-496-16 TCCTCGGCGGTCCAAA 54 8 SEQ ID No:39 AS-II-549-20 TATCTCTCCTCGGGTTTC AG 48 4 SEQ ID No:40 AS-II-579-20 GCAAAGAAAGCCAGAACATG 50 0 SEQ ID No:41 AS-1I-619-20 TCGCTCCACCAAGTrTTCAT 52 1 SEQ ID No:42 AS-II-626-20 GGCTAAATCGCTCCACCAAG 53 9 SEQ ID No:43 AS-n-634-20 AACTTCITGGCTAAATCGCT 48 0 SEQ ID No:44 AS-II-667-20 GAAGCCATAGAAACAGCGGG 53 9 SEQ ID No:45 AS-II-784-20 GACACAAGGCATCGTTTCAA 50 9 SEQ ID No:46 AS-II-798-20 TCTGCCTTCTTCTTGACACA 48.0 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 -40- PCT/CA97/00540 SEQ ID No:47 AS-II-816-20 ATCCAGCGCAAGGCCCAGTC 60 9 -43 7 SEQ ID No:48 AS-II-861-20 GCAAAGGCTACAACACGTTC 50 0 -37 1 SEQ ID No:49 AS-II-890-20 AACCGGAAAAGAAAATGCCT 52.2 -40.4 SEQ ID No:50 AS-II-909-20 CAGAATATCGACGCAAAAGA 48 2 -36 5 SEQ ID No:51 AS-n-933-20 GGCATCAGTCCTCGTTTCTT 50.8 -37 7 SEQ ID No:52 AS-II-98I-20 TGT AAACCCTCATCTCTGCT 46 2 -35 0 SEQ ID No:53 AS-II-1001-20 TCAGGCAACCAAAATCACAG 51.3 -37 2 SEQ ID No:54 AS-U-1006-20 GAACATCAGGCAAGCAAAAT 49.4 -37 1 SEQ ID No:55 AS-II-1023-20 TTGTGTACCAGGTGTTTGAA 45 9 -33 9 SEQ ID No:56 AS-II-1040-20 CTCTCTCCTCCGATGGTTTG 51 1 -37 7 SEQ ID No:57 AS-n-1048-20 TTCTCTT ACTCTCTCCTCCG 45.2 -35 0 SEQ ID No:58 AS-II-1144-20 GTATTGCTTCATT AG AGTGC 41 6 -33 0 SEQ ID No:59 AS-D-1182-20 CCCAGTTCCAGC ATA AGTCT 48 4 -36 5 SEQ ID No:60 AS-II-1197-20 AAAACCTTGCTAAAACCCAG 48 3 -37 8 SEQ ID No:6I AS-II-1217-20 CAAATGGGTTCTCTACTCTG 43 7 -33 8 SEQ ID No:62 AS-II-1224-20 ATAAAGTCAAATGGGTTCTC 42.6 -34 0 SEQ ID No:63 AS-0-1254-20 TTAGTCTTTCCTTCCAGTGA 43.8 -33 9 SEQ ID No:64 AS-D-1278-20 TCGCCTACTCTCTTCTCAAA 46 8 -35 6 SEQ ID No:65 AS-n-1288-20 CCTCTGATACTCGCCTACTC 45.6 -35.1 SEQ ID No:66 AS-n-1302-20 GACATCACTCCCATCCTCTG 48 7 -35 3 SEQ ID No:67 AS-n-1335-20 GCATCCAAGGTAAAAGAATT 45 6 -36.1 SEQ ID No:68 AS-n-1338-20 TCAGCATCCAAGGTAAAAGA 47 4 -35 9 SEQ ID No:69 AS-U-1342-20 GAAGTCAGC ATCC AAGGTAA 46 7 •35.3 SEQ ID No:70 AS-n-1345-20 TTAGAAGTCAGCATCCAAGG 47 0 -35.6 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 -41 - PCT/CA97/00540 SEQ ID No:71 AS-II-1362-20 GCACATCTTCAGTTCATTTA 42.4 -32 8 SEQ ID No:72 AS-n-1364-20 GGGCACATCTTCAGTTCATT 48 9 -36.2 SEQ ID No:73 AS-n-1381-20 AAAAATCAGCCAAGTAAGGG 48 1 -38 0 SEQ ID No:74 AS-D-1390-20 ATGGAAAAAAAAAATCAGCC 48 1 -38 0 SEQ ID No:75 AS-II-1438-20 TTCATGGTGTGGCTAGTTGG 50 8 -36 8 SEQ ID No:76 AS-II-1499-20 AGGACTGGTTGTGAGGTAGC 48 1 -35.7 SEQ ID No:77 AS-II-1517-20 CCAGCACTATAAACAGACAG 42 2 -32 8 SEQ ID No:78 AS-n-1538-20 TTCTGGCAAAAGGTGATACT 46.5 -35.6 SEQ ID No:79 AS-II-1560-20 GTAAGTCACAGCCAGCCAGG 52 2 -37 8 SEQ ID No:80 AS-II-1581-20 ACTGCCATrGTCACTGCTAT 47 0 -34.9 SEQ ID No:81 AS-U-1659-20 TGGCTGTGCTGGTTAAAGGA 53 2 -38.7 SEQ ID No:82 AS-H-1666-20 TTTTAACTGGCTGTGCTGGT 50 0 -37 2 SEQ ID No:83 AS-D-1700-20 ATTAAAATCTGCGTTGAAGC 46 8 -36 6 SEQ ID No:84 AS-II-1768-20 TATCGCCGCCGTGAGTACAA 56 5 -40.9 SEQ ID No:85 AS-II-1773-20 GCTATTATCGCCGCCGTGAG 57.1 -42 6 SEQ ID No:86 AS-II-1775-12 ATCGCCGCCGTG 42 9 -29.5 SEQ ID No:87 AS-U-1790-20 GAAACCAAATAAATCAAGCT 43 4 -34 9 SEQ ID No:88 AS-n-1819-20 TTAGTGGTCAGGAGAATGTA 41 7 -32.5 SEQ ID No:89 AS-n-1976-20 TGGCACCAACTGACTAATAT 44.5 -34 2 SEQ ID No:90 AS-n-1989-20 CCTGTCTTCTATCTGGCACC 48 6 -36.2 SEQ ID No:91 AS-n-2009-20 GCCACAGGATAAAAACACAA 47.7 -35 9 SEQ ID No:92 AS-H-2026-20 CCCAGGACACTACACAAGCC 51 8 -37 5 SEQ ID No:93 AS-n-2044-20 TCAGAGGGGGCAGAGAATCC 55 4 -40 2 SEQ ID No:94 AS-H-2067-20 TCCTTTATCCCACAACACTC 46 3 -35.0 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 -42- PCT/C A97/00540 SEQ ID No:95 AS-II-2083-20 CCTTGCCCTGAGAGATTCCT 52 3 39.0 SEQ ID No:96 AS-I1-2083-20 CsCTsTGsCCsCTsGAsGAsGAsTTsCCsT 52 3 •39 0 SEQ ID No:97 AS-II-2128-20 GGCCCAGATCACCCCTAAAT 54 3 -409 SEQ ID No:98 AS-n-2151-20 AAACGGCTTCTCACACATAT 46 3 35 4 SEQ ID No:99 AS-n-2164-20 GAGAAATAAAATGAAACGGC 46.2 •36 6 SEQ ID No:100 AS-n-2182-20 CGTTGAGGAAAATACAGTGA 45 1 -34.3 SEQ ID No: 101 AS-II-2229A-20 GCTCCC AC AT ATG AAA ACTC 46 1 -35.2 SEQ ID No: 102 AS-II-2372-20 CACACAACCTACTTACACCA 42 7 -32.3 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 -43- PCT/CA97/00540 Footnotes for Table 7 Name includes the following AS = antisense; II =R2 The first number indicates the first nucleotide position in the R2 mRNA sequence. The second number indicates the length of the sequence segment. The sequence AS-II-2229A shown in the Table and the sequence AS-II-2229B described m the text are alternate sequences, with 2229A chosen from the version of R2 in GENBANK (submitted by Pavloff) and 2229B chosen from the version published by Pavloff et al., J DNA Sequencing and Mapping, 2:227-234,1992. Sequences were fully thioated unless partial thioation is indicated (s) 1TM°C = Melting temperature of oligonucleotide duplex formed 2dG = Free energy values for ohgonucleotide-complement dimer formation In addition to the above analysis, estimates of potential dimer formation (D), potential self-complementary interactions (H), and the potential to bind to sequences in the R2 message other than the target sequence (B) were obtained. Analysis and estimates described above were obtained by using the computer modelling program OLIGO Pnmer Analysis Software, Version 3 4 (distributed by National Biosciences) The program allows the determination of Tm°C and dG values, and also provides a qualitative estimation of the D, H and B parameters indicating "no potential", "some potential" or essentially "complete potential" In choosing the oligonucleotide sequences we gave high priority to sequences that exhibited high Tm°C and dG values, which are important for tight binding of antisense molecules to their complementary strands, and high priority to antisense sequences that had estimates of no potential in D, H and B Of the three categories (D,H,B) the most important ones were D and H, since B (1 e. binding to other regions of the R2 mRNA in addition to the precise target sequence) may enhance rather than compromise oligonucleotide activity Most of the sequences shown in Table 7 had no potential in the D and H categories Some sequences exhibited "some potential" in D or H and were later found in tumor cell growth inhibition studies to be effective (Table 12) and therefore were also included in Table 7 We found that this approach to choosing antisense oligonucleotide inhibitors was extremely effective, since the vast majority of the chosen sequences exhibited anti-tumor properties as shown in Table 12. Printed from Mimosa WO 98/05769 -44- PCT/CA97/00540 TABLE8 TREATMENT WITH AN R2 ANTISENSE CONSTRUCTS CONSTRUCT CONC COLONY FORMING INHIBITION OF (Jim) MDA435 AS-II-2229B-20 0 02 25% 0 03 56% 0 05 78% 0.10 94% 0 20 99% Printed from Mimosa WO 98/05769 PCT/CA97/00540 -45- TABLE 9 SYNERGISTIC EFFECT OF AS-II-2229B-20 ANTISENSE CONSTRUCT Cells Drug Drug AS-II-2229B-20a Cone 002_M Hela S3 PALAa 20|iM - PALA 20pM + Hela S3 MTXa 40pM . MTX 60|iM - MTX 40(iM + MTX 60(iM + Hela ImM PALA 20pM - PALA 30|iM - PALA 20(tM + PALA 30|iM + Hela ImM MTX 40pM . MTX 60jiM - MTX 40nM + MTX 60 pM + Relative Colony Forming Efficiency0 350 ±50 90± 10 118+32 116 ±13 25± 5 0 377 ±21 311+95 108 ±75 101 ±2 0 28 ±10 12 ±05 65 ±5 5 35 ±0.5 a PALA = N-(phosphonacetyl)-L-aspartate a MTX = methotrexate b - = no treatment b + = treatment provided c The values are the average of two experiments. Printed from Mimosa WO 98/05769 - 46 - PCT/CA97/00540 TABLE 10 REDUCED COLONY FORMING EFFIOENCY FOLLOWING TREATMENT WITH Rl ANTISENSE CONSTRUCT CELL LINE: Hela S3 Conf. AS-I-1395-20a % Inhib 0 0.2 nM 75% (Exp 1) 0 2 pM 77% (Exp 2) CELL LINE Hela ImM Cone AS-I-1395-20a % Inhih. 0 0 01 |jM 0 005 pM 30% 0 10 nM 60% CELL LINE Mouse SC2 Conr A S-I-1395-208 % Inhib 0 0 2 pM 76% Printed from Mimosa WO 98/05769 PCT/CA97/00540 -47-TABLE 11 ANTISENSE SEQUENCES DESIGNED TO TARGET THE Rl MESSAGE SEQ ID No: Name Sequence 5' - 3' Tm°C dG kDa/mol SEQ ID No:104 AS-I-35-20 GIT CCA GCC AGA CAG CAC TT 517 -37.3 SEQ ID No:105 AS-I-37-20 GAG TTC CAG CCA GAC AGC AC 52 0 -37 0 SEQ ID No:106 AS-1-85-20 CAG ACT GGG AAG GGT TAG GT 49 7 -37.5 SEQ ID No:107 AS-I-91-20 AGG TG A CAG AGT GGG AAG GG 52.7 -38.2 SEQ ID No:108 AS-I-129-20 GAC TGG ACT GCG GCT CTA AA 521 -38 3 SEQ ID No:109 AS-I-203-20 ATG ACT CGT TCT TGG CGG CC 58 6 -42 4 SEQ ID No:110 AS-I-239-20 CAA AGC TTC TGG ATT CCA GA 49.6 -371 SEQ ID No:lll AS-I-287-20 TTC ATG GTG ATC TG A GCA GG 50 6 -36.2 SEQ ID No:112 AS-I-300-20 GCC TIG GAT TAC ITT CAT GG 48 9 -37.3 SEQ ID No:113 AS-I-348-20 TTC AGC AGC CAA AGT ATC TA 45 4 -34.9 SEQ ID No:114 AS-I-395-20 GCC AGG ATA GCA TAG TCA GG 48 9 -36 9 SEQ ID No-115 AS-I-439-20 CTTTCTTTGTTTCTTTGT GC 445 -34.6 SEQ ID No:116 AS-I-504-20 GGG AGA GTG TTT GCC ATT AT 48 2 -36.7 SEQ ID No:117 AS-I-520-20 TTG ACT TGG CCA CCA TGG GA 58 2 -40 8 SEQ ID No:118 AS-I-540-20 GGC CAG AAC AAT ATC CAA TG 49 5 -37 2 SEQ ID No:119 AS-I-556-20 TCA GGC GAT CTT TAT TGG CC 542 -40.5 SEQ ID No:120 AS-I-635-20 TTC AAC AAA TAA GAC CGC TC 47.2 -361 SEQ ID No:121 AS-I-658-20 TIT CAG CCA CTT TTC CAT TG 50 3 -37 5 SEQ ID No:122 AS-I-662-20 GGT CTT TCA GCC ACT TTT CC 50 4 -37.9 SEQ ID No:123 AS-I-782-20 TTG AAG AGA GTG GGC GAA GC 54.4 -39.6 SEQ ID No:124 AS-I-786-20 AGC ATT GAA GAG AGT GGG CG 54 3 -39 5 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 -48- PCT/CA97/00540 SEQ ID No:125 AS-I-809-20 GAA AGTTGC GGG CGG TTG GT 60.6 -44 3 SEQ ID No:126 AS-I-843-20 GCT GTC ATC TTT CAT ACT CA 41 9 -32.2 SEQ ID No:127 AS-I-908-20 CCA ATT CCT CCA GCA GAC TT 508 -37 8 SEQ ID No:128 AS-I-923-20 CAA CTC ACA GCA ACA CCA AT 48.1 -34 8 SEQ ID No:129 AS-I-932-20 GCC CGA ATA CAA CTC ACA GC 52 2 -38.2 SEQ ID No:130 AS-I-967-20 AAT TGC CAT TAG TCC CAG CA 52.2 -38.8 SEQ ID No:131 AS-I-1051-20 ATG CCC CAG GAC GCT TGT TC 58 5 -42.2 SEQ ID No:132 AS-I-1074-20 CCA AGG CTC CAG GTA AAT AG 48 4 -37 6 SEQ ID No:133 AS-I-1134-20 ACG CTG CTC TTC CTT TCC TG 53 7 -39.6 SEQ ID No:134 AS-1-1162-20 TCC AAA GAG CAA AGA AAA G A 47 0 -36.1 SEQ ID No:135 AS-I-1258-20 CCT CTC CCC AAA CCT CAT CC 547 -40 2 SEQ ID No:136 AS-I-1311-20 AAC TIT GCG GAC ACG ACC TT 53 7 -39 5 SEQ ID No:137 AS-I-1370-20 GGG GTG CCT GIT TCC GTC TG 58 9 -42.0 SEQ ID No:138 AS-I-1418-20 TTC TGC TGG TTG CTC TTT CG 53 1 -38 7 SEQ ID No:139 AS-I-1421-20 AGG TTC TGC TGG TTG CTC TT 50.6 -37 6 SEQ ID No:140 AS-I-1513-20 GGG CCA GGG AAG CCA AAT TA 57 6 -43.4 SEQ ID No:141 AS-I-1662-20 GGG GCG ATG GCG TIT ATT TG 58 8 -44 0 SEQ ID No:142 AS-I-1666-20 CAA TGG GGC GAT GGC GTTTA 601 -44 0 SEQ ID No:143 AS-I-1785-20 TTC CAG AGC ACC ATA ATA AA 45.1 -35.1 SEQ ID No:144 AS-I-1818-20 TGG GCC CTG CTC CTT GGC AA 64.3 -45.7 SEQ ID No:145 AS-I-1970-20 GGC ATC GGG GCA ATA AGT AA 541 -41 0 SEQ ID No:146 AS-I-1976-20 GCT GTA GGC ATC GGG GCA AT 58.5 -42 9 SEQ ID No:147 AS-I-2119-20 CAT GCC ATA GGC CCC GCT CG 64.0 -46.4 SEQ ID No:148 AS-I-2198-20 AGT TGC TTC AGG TCA TCA GG 49.0 -36 0 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 -49- PCT/CA97/00540 SEQ ID No:149 AS-I-2251-20 CAG CTG CCA TCT TGA GAA CA 511 -36.6 SEQ ID No:150 AS-I-2304-20 CTC AGC AAT GTG GAT GTT CA 48 9 -35 0 SEQ ID No:151 AS-I-2364-20 AGT CTT CAA ACC CTG CTT CC 50 0 -37 6 SEQ ID No:152 AS-I-2370-20 CAT CCC AGT CTT CAA ACC CT 504 -37 5 SEQ ID No:153 AS-I-2414-20 GTG AAC TGG ATT GG A TTAGC 46.1 -35 2 SEQ ID No:154 AS-I-2491-20 TGG CTG CTG TGT TCC TCT CC 55 0 -38 8 SEQ ID No:155 AS-I-2556-20 CTT CCA AGT CTT TCC TCA GG 48 0 -36 4 SEQ ID No:156 AS-I-2629-20 TAC CAC CTC AAG CAA ACC CA 52 9 -38 4 SEQ ID No:157 AS-I-2650-20 CAA CAG GGT CCA GCA AAG CC 56 8 -40.9 SEQ ID No:158 AS-I-2769-20 TCC GTT TTT TIT TTC TTTTT 46 2 -37.5 SEQ ID No:159 AS-I-2863-20 TGC TAA ATG GGT GAT GAA AC 475 -35.8 SEQ ID No:160 AS-I-2922-20 CCC ACC AGT CAA AGC AGT AA 50.2 -36 9 SEQ ID No:161 AS-I-2594-20 CTC AAG AAG TAG TTT GGC TA-3' 41 6 -33 2 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 -50- PCT/CA97/00540 Footnotes for Table 11 Name includes the following: AS = antisense I = Rl The first number indicates the first nucleotide position m the Rl mRNA sequence. The second number indicates the length of the sequence segment. iTm°C = Melting temperature of oligonucleotide duplex formed. 2dG = Free energy value for oligonucleotide-complement dimer formation. In addition to the above analysis, estimates of potential dimer formation (D), potential self-complementary interactions (H), and the potential to bind to sequences in the Rl message other than the target sequence (B), were obtained. Analyses were performed as described in the Footnote to Table 7, and criteria used to select the sequences shown in Table 11 were as indicated m the Footnote to Table 7. Printed from Mimosa * o Table 12: Reduced Relative Colony Forming Efficiency or Human Tumor Cells Following Treatment with 0.2 fiM of Various | Antisense Oligodeoxyribonucleotide Phosphorothioates Targeting the R2 Message, Expressed As % Inhibition 13 2 Name (Re) T24 HCT116 AS49 MDA-MB-231 MIA PaCa-2 PC-3 HepG2 Hela S3 T-47D HS96 Colo320 AS-11-6-20 73 85 ND ND 88.4 95 15 89 21 97 89 ND ND ND ND AS-U-13-20* 18 99 6.95 32 3 45 45 ND 52 38 24 11 19 85 15 33 19.68 ND AS-U-14-20 77 59 ND ND 91 24 47 93 92.76 88 4 ND ND ND ND AS-II-16-18 25.74 78 57 81 1 62 59 ND 89 48 75 89 68 7 7 13 34 5 ND AS-II-75-20* 73 42 44 4 60.08 49.3 97 38 68 25 35 4 93 01 32.95 ND ND AS-II-75-20 95 83 ND ND 95 14 52 07 83.46 97.89 ND ND ND ND AS-D-79-14 38 4 45.56 79.17 48 6 38 89 85 32 70 81 28 64 70 81 ND ND AS-U-109-20* 24 89 6 76 15.14 22 38 54 24 61 51 18 08 46.83 20.63 7 28 ND AS-U-110-20 87 78 71.69 89 38 90 92 47 51 92 06 97 14 53 98 ND ND ND AS-II-114-20 87 45 86 1 83.51 76 22 90.05 92 66 78 72 79.25 90.83 46 3 ND AS-II-127-12 50 63 54.34 69.33 38 46 53 24 79 56 71 75 86 45 37 54 ND ND AS-IM 30-20 51 94 57 98 8648 ND 82.11 74.66 94 28 ND ND ND ND AS-U-134-20 ND ND ND ND ND 77 51 ND ND ND ND ND AS-II-151-20 ND 78.09 84.28 41.64 75 38 85 68 89 58 66 75 95.89 69 12 90.12 AS-U-163-20* 5 49 29 05 37 13 22 73 9.88 7 14 18 64 45.8 981 32 08 ND AS-II-166-20 68.99 73 84 81 1 29 02 91 36 74.11 78 72 80 1 91 4 61 99 ND 2, 2 ui r H-» pi , *v a 9- V© >1 £ o WO 98/05769 PCT/CA97/00540 -52- TABLE12 (continued) © rj o "© U ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND H596 24 53 26 15 49 6 ND ND 0B1 ND 12.67 ND ND ND ND ND ND ND ON ND ND T-47D 94 52 96 63 98.53 ND ND 27 09 ND ND ND ND j a ND ON ND ND ON ND ND Hela S3 19.38 76 67 90 87 ON 66 96 65.18 41 79 57 63 ND ND 45 69 ND 58 37 ND ND 60 £9 ND ND HepG2 53 44 57 86 59 32 99 55 ND CM 97 14 65 89 96 39 ND 95 78 60.64 84 79 26 23 30 89 97 89 73 04 ND U Om 4 37 73.12 65 08 87 41 9008 67 26 77 51 74 01 79.76 9008 75 78 93 25 77 08 73 96 60 94 75 65 77 66 90 7 (S MIA PaCa 29 32 70 52 70 22 87.38 95 44 ND 53 94 OS O © m 97.55 ND 34 2 26 35 17.84 55 25 36 11 65 2 92 69 97 13 MDA-MB-231 174 39 86 25 17 52 7 ND 45 37 79 14 52 1 ND ND 85 83 ND 85 83 ND ND 89 75 ND ND A549 71 51 76.57 90 55 ND 92 9 61 62 61 81 70 49 91 24 ND 61 81 ND 52.17 43.78 12 44 57 38 76 46 96 99 HCT116 ND 86 78 45 56 61 85 59 5 53 28 69 42 67 57 55 8 ND 62.81 ND ND 27 98 ND 647 67 23 ! T24 21.94 18 57 96.2 65 02 73 23 1941 90 56 30.38 ON ND 89 63 ND 84 26 22 2 11 67 90 37 67 84 69 26 Name (Re) AS-II-185-20 AS-II-189-20 AS-II-201-20 AS-II-217-20 AS-II-225-20 AS-U-253-14 AS-II-280-20 AS-11-288-12 AS-II-323-20 AS-II-344-20 AS-II-362-20 AS-ll-391-17 AS-II-404-20 AS-II-412-20 AS-II-414-20 AS-II-425-20 S ro tj- ir, < AS-II-472-20 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 PCT/CA97/00540 -53- TABLE12 (continued) © N 0 ND ON ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND o U 96SH ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND T-47D , ND 10 79 ND ND ND 66 12 ND ND 85 39 ND ON ND 1 ND j ND ND ND ND j ND C/3 "3 X 48 62 52 21 27 8 ND 31 22 67 92 48 94 ND 81 48 56 42 ND ND ND ND 26 86 69 43 ON 41.79 M o a a> X 93 37 54 8 84 04 94 28 92 02 75 89 95 78 88 23 80 89 89 46 93.08 96 08 ND ND 79 97 95 03 90.36 89 31 PC-3 80 6 88 84 ND 88 16 88 01 83 23 77 86 8 16 88 99 83 21 94 08 87 33 87 93 84 86 86 91 85.16 85 94 92 58 MIA PaCa 25.31 ND 1701 97 55 39 83 75.62 ON 85.26 62.04 34 65 93 21 97 3 66.02 46 13 35 68 ND 64.21 47.93 . "? MDA MB-2 54 78 45 8 40 13 66 89 91.08 90 21 92 44 § 33 92 83.92 S 89 98 88 2 45 96 64.89 91.48 82 17 S A549 33.85 74.45 30 57 95.49 65.67 95 14 57 76 70 3 00 m QO 70 49 78 4 95 78 81 6 78 68 53.52 74.25 71.36 61.62 HCT116 50.28 70 85 47 83 8069 ND 70 46 ND 76 9 78.09 64 46 88 22 74.2 ON ND 67 24 66 84 55.58 45 56 n H 54 23 78.48 45 46 76 68 86 3 76 79 83.52 70 48 87.23 84 72 73.91 73.5 82 07 78.57 64 84 86 3 86.11 61 49 Name (Re) AS-II-494-20 AS-II-496-16 AS-II-549-20 AS-II-579-20 AS-II-619-20 AS-II-626-20 AS-n-634-20 AS-II-667-20 AS-n-784-20 AS-II-798-20 8 oo C/5 < AS-II-861-20 AS-II-890-20 AS-II-909-20 AS-lI-933-20 AS-II-981-20 1 ea CO < AS-D-1006-20 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 PCT/CA97/00540 -54- TABLE12 (continued) Colo320 « ND ND ND ND ND ND ON ON ■ ND ND ND ON ND ND ND HS96 ND ND ND ND ON ND ON ON ON ND ON ND ND ND ND ND ND Q r- ND 64 76 ND ON 92.41 81 95 ! ND j ND ON ND ND ND ND ND ND ND ND Hela S3 ND 71 87 39 19 j 50 57 19 06 82 68 ND 148 7 64 ND ND ND ND ND ND ND HepG2 ND j 77 78 77 56 92 17 92 23 76.32 ND 76 05 83 28 ND 80.41 ND 72 98 8041 82.2 75 42 80 41 PC-3 87 63 40 08 84 38 CN O OO 92 76 81 03 9908 73.31 85 31 93.55 79 01 90 04 93 75 89 84 89 58 77 38 MLA PaCa-2 42 82 43.52 33.4 46 89 84 72 70 68 V) 00 en 17 22 3 32 57.05 80.71 23 86 78.78 79 17 73 77 77 39 44 14 MDA-MB-231 ND ON | 66.88 ND ND 50 35 ND ND ND 90 68 ND 92 2 ND ND ND 94 11 70 22 A549 34.52 85.82 65.67 1819 80 06 89 15 91.49 55 06 36 74 ND 61.04 93.17 78 59 63 93 65 67 73 1 83 9 HCT116 ON 70 08 42 63 54 25 88 13 84 85 89 89 41 78 28.54 ND 66 43 71.98 67 87 74.73 73.74 65.7 78 47 T24 58 26 59.49 40.32 62 9 94 51 90 3 66.36 OO CO 41 53 65.42 56 75 70 56 59 95 63 16 59.76 51.26 ND Name (Re) AS-IM023-20 o CN ci 3 ea CO < AS-IM 048-20 AS-n-1144-20 AS-C-1182-20 AS-n-1197-20 AS-H-1217-20 AS-U-1224-20 AS-n-1254-20 AS-n-1278-20 AS-II-1288-20 AS-II-1302-20 AS-II-1335-20 AS-U-1338-20 AS-U-1342-20 AS-U-1345-20 ASH-1362-20 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 PCT/CA97/00540 -55- TABLE12 (continued) © fN o e U ND ND , ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND H596 ND 45 28 ND ON 78 3 __ ON 8 71.16 ND ND ND ND ON 66 04 186 § ND T-47D ND 93 62 ND ND 96 67 ON ND 9 98 ND ND 1 1 ■" " ND ND 1 ND 84 38 ND ON ON Hela S3 ND 80 53 ND ND 87 56 ND ND 82 16 ND s ND ND ND 80 27 68 61 ND ND N O Ok V X ND 62 34 rs o OS ND [77 5 ND 96 84 73 63 93 07 ON ND ND ND ON OO 43 97 ON 83 89 PC-3 79 01 75 76.76 83 96 77 28 91.75 84 71 78 37 85 83 81.56 86 06 90 63 86 36 70 04 78 17 00 00 85.46 MIA PaCa-2 59 34 73.77 82 77 42.13 | 81.17 ND 50 62 00 00 24.07 42 54 49.72 34.52 60 36 45 68 27 78 ND 20 02 MDA-MB-231 93 87 44 41 66 89 69 06 41 26 ND ND 46.15 ND 42 86 26 71 30 75 ! 91 56 44 76 48 6 ND ! 89 06 A549 95 59 86.02 8831 51 27 87 8 ND 90.34 84 19 90 55 54.7 54.82 77.28 86.8 70.11 63.55 ND ND IHCT116 77 29 89.48 62 18 ND 83 01 ON 51.28 00 V") 00 66 85 ND ND ND ND 75 58 54 44 ND ND T24 1 66 59 71 37 61.13 43.7 O0 rs 06 ND 67 29 32.49 68.22 74 09 71 71 70 94 74 56 15.19 85.54 ND 53 74 Name (Re) AS-H-1364-20 AS-n-1381-20 AS-H-1390-20 o <N 00 4 < AS-II-1499-20 AS-n-1517-20 AS-H-1538-20 AS-H-1560-20 AS-U-1581-20 AS-U-1659-20 AS-H-1666-20 AS-U-1700-20 AS-H-1768-20 AS-U-1773-20 AS-H-1775-12 AS-U-1790-20 AS-U-1819-20 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 PCT/CA97/00540 -56- TABLE12 (continued) Colo320 ND ND ND ND ND ND OO oo 52.54 ND ND ND ND ND ND H596 ND I 56.33 ND 84 16 ND ND 82 98 49.87 ND ND ND ND ND ND T-47D ND 90.83 ND 93 41 ND ND 89.32 48 61 ON ND ND ND CM OO 00 m ND Hela S3 ND 61 41 ND 83 79 24.55 24 88 84 58 96 9 ON ND ND ND 63 15 ND HepG2 ND 80 7 96.99 94 54 62 35 90 36 93 64 46 14 ND ND 93.07 95.48 73 26 ON PC-3 NO OO OO 77 38 83 36 95.29 74 48 85 94 ND 82 03 88.89 84 86 83.36 85 91 89 58 90 98 MIA PaCa-2 ND 70 22 93 74 87 65 24 48 41 08 r- 39 23 62 15 89 36 56.43 33 61 94 36 ! MDA-MB-231 89 6 54 9 96 25 VLL II 61 49.68 68 88 ND 91 3 95 14 69 03 84 95 ND 60 06 A549 ON 83.9 93 32 OO OO 00 25.55 64.71 90.65 31 73 87 31 79 19 91 15 90.97 69.72 9641 1 HCT1I6 ND 78 47 69.92 81 47 49 34 47.16 87.46 41 16 ND ON 50 08 77 66 | 93 01 64 7 H | ND r- 00 3 95 46 53.63 49 6 82 43 9 52 83.74 79 83 61 84 67.76 50 34 61.13 Name (Re) AS-II-1976-20 AS-H-1989-20 AS-11-2009-20 AS-U-2026-20 AS-U-2044-20 AS-n-2067-20 AS-U-2083-20 AS-U-2083-20* AS-II-2128-20 AS-n-2151-20 AS-U-2164-20 AS-U-2182-20 AS-II-2229A-20 AS-n-2372-20 SUBSTITUTE SHEET (RULE 26) Printed from Mimosa WO 98/05769 PCT/CA97/00540 -57- Legend to Table 12 - The antisense oligonucleotides were fully thioated unless indicated (*), as described m Table 7. - The values for relative colony-forming efficiencies are averages obtained from 2-8 determinations - ND = not determined. - The various cell lines were obtained from the American Type Culture Collection, Rockville, Maryland Information about these human cancer cells. T24 = bladder cell carcinoma HCT116 = colon cell carcinoma A549 = lung cell carcinoma MDA-MB-231 = breast cell adenocarcinoma MIA PaCa-2 = pancreatic cell carcinoma PC-3 = prostate cell adenocarcinoma HepG2 = hepatocellular carcinoma HelaS3 = cells isolated from a carcinoma of the cervix T-47D = breast ductal carcinoma H596 = lung adenosquamous carcinoma cells Colo320 = colon cell adenocarcinoma Printed from Mimosa WO 98/05769 PCT/CA97/00540 -58- TABLE 13 Metastatic Characteristics of r-3 Mouse IOTV2 Tumor Cells in Syngeneic Mice Following Treatment with the Antisense Oligonucleotides, AS-II-626-20 Oligonucleotide * Treatment Frequency of Mice with Tumors Number of Lung Tumors (mean ± SE) none 4/4 6.0 ± 1.58 0.2 nM 1/4 0 25 ± 0 25 *105 cells either treated for 4 hours with lipofectin without oligonucleotide supplement (none) or with lipofectin containing 0.2 (0.M AS-II-626-20, were injected intravenously (tail vein) into C3H/HeN syngeneic mice and lung tumors were analyzed as previously described (Damen, J.E., Greenberg, A.H. and Wright, J.A. 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Hydroxyurea and related compounds In R S Gupta (ed ), Drug Resistance in Mammalian Cells, Boca Raton, FL, CRC Press, Inc, 15-27 15 Wright et al, 1990A Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase and the significance to DNA synthesis Biochem Cell Biol. 68 1364-1371 Wright et al, 1993 Transforming growth factor [3 and fibroblast growth factor as promoters of tumor progression to malignancy. Cnt. Rev Oncogen 4:473-492. Yakubov et al, 1989 PNAS USA 86 6454 Yin et al., 1992. Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles Cell 70 937-948 Printed from Mimosa INTELLECTUAL PROPERTY OFFICE OF N.Z. - 7 AUG 2001 - 65 - received </div>

Claims

<div class="application article clearfix printTableText" id="claims"> Claims:

1. An antisense oligonucleotide comprising at least seven to about thirty-five nucleotides and a sequence complementary to a sequence of a mammalian ribonucleotide reductase gene, or an analogue thereof, wherein said oligonucleotide, or analogue thereof, is capable of inhibiting the proliferation of neoplastic cells.

2. The oligonucleotide according to claim 1 which is complementary to a mammalian ribonucleotide reductase Rl gene, or an analogue thereof.

3. The oligonucleotide according to claim 1 which is complementary to a mammalian ribonucleotide reductase R2 gene, or an analogue thereof.

4. The oligonucleotide according to claim 3 wherein the oligonucleotide comprises a nucleic acid sequence as set forth in one or more of SEQ ID NOS: 1 to 102, or an analogue thereof.

5. The oligonucleotide according to claim 3 wherein the oligonucleotide comprises a nucleic acid sequence as set forth in SEQ ID NOS: 1, 2, 12, 16, 18, 21, 25, 29, 34, 42, 44,45, 46, 52, 53, 59, 60, 64, 65, 66, 68, 69, 70, 72, 73, 74, 76, 78, 79, 80, 90, 91, 92, 96, 99, 100, or 102, or an analogue thereof.

6. The oligonucleotide according to claim 5 wherein the oligonucleotide comprises the nucleic acid sequence set forth in SEQ ID NO: 42, or an analogue thereof.

7. The oligonucleotide according to claim 2 wherein the oligonucleotide comprises a nucleic acid sequence as set forth in one or more of SEQ ID NOS 103 to 161, or an analogue thereof. 66- ITZL' ICTU/ L P^OPITRTY OFFICE OF N.Z. - 7 AOS 2001 RECEIVED

8. The oligonucleotide according to claim 7 wherein the oligonucleotide comprises a nucleic acid sequence as set forth in SEQ.ID NO 103, or an analogue thereof.

9. The oligonucleotide according to any one of claims 1 to 8 wherein the oligonucleotide, or an analogue thereof, exhibits reduced dimer formation and reduced self-complementary interactions.

10. A pharmaceutical composition for inhibiting neoplastic cell proliferation comprising at least one antisense oligonucleotide, or an analogue thereof, according to any one of claims 1 to 9, in admixture with a physiologically acceptable carrier or diluent.

11. A pharmaceutical composition for increasing the sensitivity of neoplastic cells to a chemotherapeutic drug comprising at least one antisense oligonucleotide, or an analogue thereof, according to any one of claims 1 to 9 in admixture with a physiologically acceptable carrier or diluent.

12. A pharmaceutical composition for inhibiting the growth of neoplastic cells that are resistant to a chemotherapeutic drug comprising at least one antisense oligonucleotide, or an analogue thereof, according to any one of claims 1 to 9 in admixture with a physiologically acceptable carrier or diluent.

13. The pharmaceutical composition according to claim 11 or 12, wherein said chemotherapeutic drug is PALA, MTX or hydroxyurea.

14. A pharmaceutical composition for reducing metastasis of neoplastic cells, comprising at least one antisense oligonucleotide, or an analogue thereof, according to any one of claims 1 to 9, in admixture with a physiologically acceptable carrier or diluent. INTELLECTUAL PROPERTY OFFICE OF N.Z. - 7 AUG 2001 _67_ received

15. A use of at least one antisense oligonucleotide, or an analogue thereof, according to any one of claims 1 to 9 to prepare a medicament for inhibiting neoplastic cell proliferation.

16. A use of at least one antisense oligonucleotide, or an analogue thereof, according to any one of claims 1 to 9 to prepare a medicament for increasing the sensitivity of neoplastic cells to a chemotherapeutic drug.

17. A use of at least one antisense oligonucleotide, or an analogue thereof, according to any one of claims 1 to 9 to prepare a medicament for inhibiting the growth of neoplastic cells that are resistant to a chemotherapeutic drug.

18. The use according to claim 16 or 17, wherein said chemotherapeutic drug is PALA, MTX or hydroxyurea.

19. A use of at least one antisense oligonucleotide, or an analogue thereof, according to any one of claims 1 to 9 to prepare a medicament for reducing metastasis of neoplastic cells.

20. A DNA comprising a transcription initiation region and encoding at least one antisense oligonucleotide according to any one of claims 1 to 9.

21. A vector comprising the DNA according to claim 20.

22. A use of the vector according to claim 21 to prepare a medicament for inhibiting neoplastic cell proliferation. 68 INTELLECTUAL PROPERTY OFFICE OF N.Z. - 7 AUG 2001 received

23. A use of the vector according to claim 21 to prepare a medicament for increasing the sensitivity of neoplastic cells to a chemotherapeutic drug.

24. A use of the vector according to claim 21 to prepare a medicament for inhibiting the growth of neoplastic cells that are resistant to a chemotherapeutic drug.

25. The use according to claim 23 or 24, wherein said chemotherapeutic drug is PALA, MTX or hydroxyurea.

26. A use of the vector according to claim 21 to prepare a medicament for reducing metastasis of neoplastic cells.

27. A method of evaluating if a compound inhibits transcription or translation of a ribonucleotide reductase gene and thereby affects cell proliferation by transfecting a cell with an expression vector comprising a recombinant molecule comprising a nucleic acid sequence encoding ribonucleotide reductase, and the necessary elements for the transcription or translation of the nucleic acid; administering a test compound; and comparing the level of expression of the ribonucleotide reductase with the level obtained with a control in the absence of the test compound.

28. A method of evaluating a compound for its ability to regulate a Ras signalling pathway by assaying for an agonist or antagonist of the interaction of R2 and Raf-1 and/or Rac-1 by providing a reaction mixture containing R2 and Raf-1 and/or Rac-1 under conditions which permit the interaction of R2 and Raf-1 and/or Rac-1, in the presence of a test compound; detecting the formation of complexes between R2 and Raf-1 and/or Rac-1 or activation of a Ras signalling pathway; and comparing to a control reaction in the absense of the test substance, wherein lower levels of complexes or activation in the reaction mixture indicate that the test compound intellectual PROPERTY OFFICE OF N.Z ~ 7 AUG 2001 Received 69- interferes with the interaction of R2 and Raf-1 and/or Rac-1, and higher levels indicate that the test compound enhances the interaction of R2 and Raf-1 and/or Rac-1. </div>